DOCSLIB.ORG

The Regulation of Self-Renewal in Normal Human Urothelial Cells

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Regulation of Self-Renewal in Normal Human Urothelial Cells The Regulation of Self-Renewal in Normal Human Urothelial Cells Lisa A. Kirkwood PhD University of York Department of Biology April 2012 Abstract The urinary tract is lined by a mitotically-quiescent, but highly regenerative epithelium, the urothelium. The mechanisms regulating urothelial regeneration are incompletely understood although autocrine stimulation of the Epidermal Growth Factor Receptor (EGFR) signalling pathway has been implicated. The hypothesis developed in this thesis is that urothelial homeostasis is regulated through resolution of interactive signal transduction networks downstream of local environmental cues, such as cell:cell contact. Here, canonical Wnt signalling was examined as a candidate key pathway due to the pivotal role of β-catenin in both nuclear transcription and intercellular adherens junctions. Normal human urothelial (NHU) cells isolated from surgical biopsies were grown as finite cell lines in monolayer culture. mRNA analysis from proliferating cultures inferred all components for a functional autocrine-activated canonical Wnt cascade were present. In proliferating cells, β-catenin was nuclear and Axin2 expression provided an objective hallmark of β-catenin/TCF transcription factor activity. This endogenous activity was not mediated by Wnt receptor activation, as Wnt ligand was produced in inactive (non-palmitylated) form in serum-free culture, but instead -catenin activation was driven via EGFR- mediated phosphorylation of GSK3 and inhibition of the β-catenin destruction complex. In quiescent, contact–inhibited cultures, β-catenin was seen to re- localise to the adherens junctions and GSK3β activity was re-established. Knock-down of β-catenin using RNA interference led to significant changes in p-ERK and p-AKT activity as well as an increase in E-cadherin protein expression. The results presented in this thesis identifies β-catenin as a central component of a bi-directional feedback loop between growth factor-mediated cell signalling and cell:cell contact and provides preliminary evidence that de- regulation of the mechanisms that control β-catenin regulation and EGFR signalling are important in neoplastic growth. 2 Contents 1 Introduction ...................................................................................... 21 1.1 The urinary system ....................................................................... 21 1.2 The kidneys ................................................................................. 22 1.2.1 The ureters ............................................................................ 23 1.2.2 The bladder ........................................................................... 24 1.3 The Human Urothelium ................................................................. 26 1.3.1 Tissue Architecture ................................................................. 26 1.3.2 Origins of the human urothelium .............................................. 26 1.3.3 Characteristics of the urothelium in situ .................................... 28 1.3.4 Function of the urothelium ...................................................... 30 1.4 Urothelial tissue homeostasis ......................................................... 31 1.5 Cancer: When tissue homeostasis goes wrong ................................. 33 1.5.1 Bladder Cancer ....................................................................... 34 1.6 Factors regulating human urothelial tissue homeostasis .................... 38 1.6.1 Cell signalling ......................................................................... 38 1.6.2 Cell environment .................................................................... 43 1.7 The canonical Wnt pathway ........................................................... 44 1.8 Wnt/β-catenin signalling in the bladder ........................................... 46 1.9 Urothelial cells in vitro ................................................................... 48 1.9.1 Isolation and propagation ........................................................ 48 1.9.2 NHU cell phenotype ................................................................ 48 1.9.3 NHU growth and proliferation .................................................. 49 1.9.4 NHU cell differentiation ........................................................... 50 1.10 Thesis Aims ................................................................................ 51 3 2 Materials and Methods ........................................................................ 53 2.1 General ....................................................................................... 53 2.2 Suppliers ..................................................................................... 53 2.3 Dissection equipment, glassware and disposable plasticware ............. 53 2.4 Stock solutions ............................................................................. 53 2.5 Reagents ..................................................................................... 54 2.5.1 Antibodies .............................................................................. 54 2.5.2 Agonists/Antagonists ............................................................... 54 2.6 Tissue Culture .............................................................................. 57 2.6.1 General ................................................................................. 57 2.6.2 Primary Urothelial Cell Culture .................................................. 57 2.6.3 Differentiation of human urothelial cell lines .............................. 59 2.6.4 Culture of established carcinoma cell lines ................................. 60 2.6.5 Mycoplasma testing ................................................................ 62 2.6.6 Genotyping ............................................................................ 63 2.7 Molecular Biology ......................................................................... 64 2.7.1 General ................................................................................. 64 2.7.2 Plasticware ............................................................................ 65 2.7.3 Plasmids ................................................................................ 65 2.7.4 Amplification of plasmid DNA ................................................... 66 2.7.5 Molecular cloning .................................................................... 68 2.7.6 Genetic manipulation of NHU cells ............................................ 70 2.8 Gene Expression Analysis .............................................................. 73 2.8.1 General ................................................................................. 73 2.8.2 RNA Extraction ....................................................................... 73 4 2.8.3 DNase treatment of RNA ......................................................... 74 2.8.4 RNA precipitation using sodium acetate .................................... 74 2.8.5 Reverse Transcriptase Polymerase Chain Reaction ..................... 75 2.8.6 First-strand cDNA synthesis -Reverse transcription (RT) .............. 75 2.8.7 Primer design for PCR ............................................................. 76 2.8.8 PCR primer testing .................................................................. 76 2.8.9 PCR ....................................................................................... 77 2.8.10 Quantitative Polymerase Chain Reaction (RTqPCR) using SYBR® - Green I technology ............................................................................ 77 2.8.11 Primer design for qPCR ........................................................... 78 2.8.12 RTqPCR primer testing ............................................................ 78 2.8.13 RTqPCR ................................................................................. 78 2.8.14 AffymetrixTM analysis ............................................................... 80 2.9 Protein Analysis ............................................................................ 80 2.9.1 Indirect immunofluorescent labelling of cultured cells ................. 80 2.9.2 Western blotting ..................................................................... 82 2.10 Cell based assays ...................................................................... 85 2.10.1 Cell viability assay ................................................................... 85 2.10.2 Luciferase Reporter assays ...................................................... 86 2.10.3 Flow Cytometry ...................................................................... 92 2.11 Statistical analysis ...................................................................... 92 3 Screening for components of the canonical Wnt pathway in Normal Human Urothelial cells ......................................................................................... 93 3.1 Aims ........................................................................................... 93 3.2 Experimental Design ..................................................................... 94 5 3.3 Results ........................................................................................ 98 3.3.1 Quality control assessment ...................................................... 98 3.3.2 Assessment of the proliferation signature ................................ 100 3.3.3 Assessment of differentiation ................................................. 102 3.3.4 Ontology analysis ................................................................
Load more

Recommended publications
  • ISSN: 2320-5407 Int. J. Adv. Res. 8(06), 797-810
    ISSN: 2320-5407 Int. J. Adv. Res. 8(06), 797-810 Journal Homepage: - www.journalijar.com Article DOI: 10.21474/IJAR01/11159 DOI URL: http://dx.doi.org/10.21474/IJAR01/11159 RESEARCH ARTICLE GROWTH FACTORS - PERIODONTAL REGENERATION: CURRENT OPINIONS IN PERIODONTOLOGY Dr. Kousain Sehar1, Dr. Nadia Irshad2, Dr. Navneet kour1, Dr. Manju Verma2 and Dr. Mir Tabish Syeed3 1. MDS Dept of Periodontology and Implantology, BRS Dental College and Hospital Sultanpur Panchkula. 2. MDS Dept of Paedontics and Preventive Dentistry, BRS Dental College and Hospital Sultanpur Panchkula. 3. MDS Dept of Paedontics and Preventive Dentistry, Swami Devi Dayal Hospital and Dental College, Barwala, Panchkula. …………………………………………………………………………………………………….... Manuscript Info Abstract ……………………. ……………………………………………………………… Manuscript History Periodontal tissue has the capacity for repair and regeneration. Received: 10 April 2020 Periodontal regeneration definition implies the formation of new bone, Final Accepted: 12 May 2020 new cementum and a functionally oriented periodontal ligament. Published: June 2020 Key words:- Periodontal, Repair, Regeneration Copy Right, IJAR, 2020,. All rights reserved. …………………………………………………………………………………………………….... Introduction:- Periodontitis, evoked by the bacterial biofilm (dental plaque) that forms around teeth, progressively destroys the periodontal tissues supporting the teeth, including the periodontal ligament, cementum, alveolar bone and gingiva1. Thus, the rationale of periodontal therapy is to eradicate the inflammation of the periodontal tissues, to arrest the destruction of soft tissue and bone caused by periodontal disease, and regenerates the lost tissue2. Periodontal surgical procedures have focused on the removal of hard and soft tissue defects by regenerating new attachment3.Periodontal wound-healing studies, however, indicate that conventional periodontal therapy most commonly results in repair rather than regeneration4, 5.
    [Show full text]
  • Diseasespecific and Inflammationindependent Stromal
    Full Length Arthritis & Rheumatism DOI 10.1002/art.37704 Disease-specific and inflammation-independent stromal alterations in spondyloarthritis synovitis Nataliya Yeremenko1,2, Troy Noordenbos1,2, Tineke Cantaert1,3, Melissa van Tok1,2, Marleen van de Sande1, Juan D. Cañete4, Paul P. Tak1,5*, Dominique Baeten1,2 1Department of Clinical Immunology and Rheumatology and 2Department of Experimental Immunology, Academic Medical Center/University of Amsterdam, the Netherlands. 3Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA. 4Department of Rheumatology, Hospital Clinic de Barcelona and IDIBAPS, Spain. 5Arthrogen B.V., Amsterdam, the Netherlands. *Currently also: GlaxoSmithKline, Stevenage, U.K. Corresponding author: Dominique Baeten, MD, PhD, Department of Clinical Immunology and Rheumatology, F4-105, Academic Medical Center/University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail: [email protected] This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/art.37704 © 2012 American College of Rheumatology Received: Apr 11, 2012; Revised: Jul 25, 2012; Accepted: Sep 06, 2012 Arthritis & Rheumatism Page 2 of 36 Abstract Objective: The molecular processes driving the distinct patterns of synovial inflammation and tissue remodelling in spondyloarthritis (SpA) versus rheumatoid arthritis (RA) remain largely unknown. Therefore, we aimed to identify novel and unsuspected disease- specific pathways in SpA by a systematic and unbiased synovial gene expression analysis. Methods: Differentially expressed genes were identified by pan-genomic microarray and confirmed by quantitative PCR and immunohistochemistry using synovial tissue biopsies of SpA (n=63), RA (n=28) and gout (n=9) patients.
    [Show full text]
  • 0.5) in Stat3∆/∆ Compared with Stat3flox/Flox
    Supplemental Table 2 Genes down-regulated (<0.5) in Stat3∆/∆ compared with Stat3flox/flox Probe ID Gene Symbol Gene Description Entrez gene ID 1460599_at Ermp1 endoplasmic reticulum metallopeptidase 1 226090 1460463_at H60c histocompatibility 60c 670558 1460431_at Gcnt1 glucosaminyl (N-acetyl) transferase 1, core 2 14537 1459979_x_at Zfp68 zinc finger protein 68 24135 1459747_at --- --- --- 1459608_at --- --- --- 1459168_at --- --- --- 1458718_at --- --- --- 1458618_at --- --- --- 1458466_at Ctsa cathepsin A 19025 1458345_s_at Colec11 collectin sub-family member 11 71693 1458046_at --- --- --- 1457769_at H60a histocompatibility 60a 15101 1457680_a_at Tmem69 transmembrane protein 69 230657 1457644_s_at Cxcl1 chemokine (C-X-C motif) ligand 1 14825 1457639_at Atp6v1h ATPase, H+ transporting, lysosomal V1 subunit H 108664 1457260_at 5730409E04Rik RIKEN cDNA 5730409E04Rik gene 230757 1457070_at --- --- --- 1456893_at --- --- --- 1456823_at Gm70 predicted gene 70 210762 1456671_at Tbrg3 transforming growth factor beta regulated gene 3 21378 1456211_at Nlrp10 NLR family, pyrin domain containing 10 244202 1455881_at Ier5l immediate early response 5-like 72500 1455576_at Rinl Ras and Rab interactor-like 320435 1455304_at Unc13c unc-13 homolog C (C. elegans) 208898 1455241_at BC037703 cDNA sequence BC037703 242125 1454866_s_at Clic6 chloride intracellular channel 6 209195 1453906_at Med13l mediator complex subunit 13-like 76199 1453522_at 6530401N04Rik RIKEN cDNA 6530401N04 gene 328092 1453354_at Gm11602 predicted gene 11602 100380944 1453234_at
    [Show full text]
  • Long, Noncoding RNA Dysregulation in Glioblastoma
    cancers Review Long, Noncoding RNA Dysregulation in Glioblastoma Patrick A. DeSouza 1,2 , Xuan Qu 1, Hao Chen 1,3, Bhuvic Patel 1 , Christopher A. Maher 2,4,5,6 and Albert H. Kim 1,6,* 1 Department of Neurological Surgery, Washington University School of Medicine in St. Louis, St. Louis, MO 63110, USA; [email protected] (P.A.D.); [email protected] (X.Q.); [email protected] (H.C.); [email protected] (B.P.) 2 Department of Internal Medicine, Washington University School of Medicine in St. Louis, St. Louis, MO 63110, USA; [email protected] 3 Department of Neuroscience, Washington University School of Medicine in St. Louis, St. Louis, MO 63110, USA 4 Department of Biomedical Engineering, Washington University School of Medicine in St. Louis, St. Louis, MO 63110, USA 5 McDonnell Genome Institute, Washington University School of Medicine in St. Louis, St. Louis, MO 63110, USA 6 Siteman Cancer Center, Washington University School of Medicine in St. Louis, St. Louis, MO 63110, USA * Correspondence: [email protected] Simple Summary: Developing effective therapies for glioblastoma (GBM), the most common primary brain cancer, remains challenging due to the heterogeneity within tumors and therapeutic resistance that drives recurrence. Noncoding RNAs are transcribed from a large proportion of the genome and remain largely unexplored in their contribution to the evolution of GBM tumors. Here, we will review the general mechanisms of long, noncoding RNAs and the current knowledge of how these impact heterogeneity and therapeutic resistance in GBM. A better understanding of the molecular drivers required for these aggressive tumors is necessary to improve the management and outcomes Citation: DeSouza, P.A.; Qu, X.; of this challenging disease.
    [Show full text]
  • Targeted Exome Sequencing Provided Comprehensive Genetic Diagnosis of Congenital Anomalies of the Kidney and Urinary Tract
    Journal of Clinical Medicine Article Targeted Exome Sequencing Provided Comprehensive Genetic Diagnosis of Congenital Anomalies of the Kidney and Urinary Tract 1,2, 3,4, 3 1,5 Yo Han Ahn y, Chung Lee y, Nayoung K. D. Kim , Eujin Park , Hee Gyung Kang 1,2,6,* , Il-Soo Ha 1,2,6, Woong-Yang Park 3,4,7 and Hae Il Cheong 1,2,6 1 Department of Pediatrics, Seoul National University College of Medicine, Seoul 03080, Korea; [email protected] (Y.H.A.); [email protected] (E.P.); [email protected] (I.-S.H.); [email protected] (H.I.C.) 2 Department of Pediatrics, Seoul National University Children’s Hospital, Seoul 03080, Korea 3 Samsung Genome Institute, Samsung Medical Center, Seoul 06351, Korea; [email protected] (C.L.); [email protected] (N.K.D.K.); [email protected] (W.-Y.P.) 4 Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul 06351, Korea 5 Department of Pediatrics, Kangnam Sacred Heart Hospital, Hallym University College of Medicine, Seoul 07441, Korea 6 Kidney Research Institute, Medical Research Center, Seoul National University College of Medicine, Seoul 03080, Korea 7 Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 16419, Korea * Correspondence: [email protected] These authors equally contributed to this article. y Received: 31 January 2020; Accepted: 8 March 2020; Published: 10 March 2020 Abstract: Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of chronic kidney disease in children.
    [Show full text]
  • Repeated BCG Treatment of Mouse Bladder Selectively Stimulates Small
    BMC Cancer BioMed Central Research article Open Access Repeated BCG treatment of mouse bladder selectively stimulates small GTPases and HLA antigens and inhibits single-spanning uroplakins Marcia R Saban1, Helen L Hellmich2, Cindy Simpson1, Carole A Davis1, Mark L Lang3, Michael A Ihnat4, Michael A O'Donnell5, Xue-Ru Wu6 and Ricardo Saban*1 Address: 1Department of Physiology, The University Oklahoma Health Sciences Center, Oklahoma City, USA, 2Department of Anesthesiology, University of Texas Medical Branch, Galveston, USA, 3Department of Microbiology and Immunology The University Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA, 4Department of Cell Biology, The University Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA, 5Department of Urology, University of Iowa, UI Hospitals and Clinics, Iowa City, Iowa 52242-1089, USA and 6Department of Urology, New York University, School of Medicine, New York, NY 10016, USA Email: Marcia R Saban - [email protected]; Helen L Hellmich - [email protected]; Cindy Simpson - [email protected]; Carole A Davis - [email protected]; Mark L Lang - [email protected]; Michael A Ihnat - [email protected]; Michael A O'Donnell - [email protected]; Xue-Ru Wu - [email protected]; Ricardo Saban* - [email protected] * Corresponding author Published: 2 November 2007 Received: 29 August 2007 Accepted: 2 November 2007 BMC Cancer 2007, 7:204 doi:10.1186/1471-2407-7-204 This article is available from: http://www.biomedcentral.com/1471-2407/7/204 © 2007 Saban et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
    [Show full text]
  • Product Description SALSA MLPA Probemix P476-A1 ZNRF3
    MRC-Holland ® Product Description version A1-02; Issued 25 September 2018 MLPA Product Description SALSA ® MLPA ® Probemix P476-A1 ZNRF3 To be used with the MLPA General Protocol. Version A1. New Product. Catalogue numbers: • P476-025R: SALSA MLPA Probemix P476 ZNRF3, 25 reactions. • P476-050R: SALSA MLPA Probemix P476 ZNRF3, 50 reactions. • P476-100R: SALSA MLPA Probemix P476 ZNRF3, 100 reactions. To be used in combination with a SALSA MLPA reagent kit, available for various number of reactions. MLPA reagent kits are either provided with FAM or Cy5.0 dye-labelled PCR primer, suitable for Applied Biosystems and Beckman capillary sequencers, respectively (see www.mlpa.com ). Certificate of Analysis: Information regarding storage conditions, quality tests, and a sample electropherogram from the current sales lot is available at www.mlpa.com . Precautions and warnings: For professional use only. Always consult the most recent product description AND the MLPA General Protocol before use: www.mlpa.com . It is the responsibility of the user to be aware of the latest scientific knowledge of the application before drawing any conclusions from findings generated with this product. General information: The SALSA MLPA Probemix P476 ZNRF3 is a research use only (RUO) assay for the detection of deletions and duplications in the ZNRF3 gene. ZNRF3 is an E3 ubiquitin-protein ligase that acts as a negative feedback regulator of Wnt signalling (Hao et al. 2012). Three independent studies show homozygous deletions of the ZNRF3 gene in 10 to 16% of adrenocortical carcinoma cases (Assié et al. 2014; Juhlin et al. 2015; Zheng et al. 2016). Moreover, in 51% of microsatellite stable colorectal cancers deletion events at the ZNRF3 locus are detected (Bond et al.
    [Show full text]
  • Identification of Active Signaling Pathways by Integrating Gene Expression and Protein Interaction Data Md Humayun Kabir1,2,3, Ralph Patrick2,4,5, Joshua W
    Kabir et al. BMC Systems Biology 2018, 12(Suppl 9):120 https://doi.org/10.1186/s12918-018-0655-x RESEARCH Open Access Identification of active signaling pathways by integrating gene expression and protein interaction data Md Humayun Kabir1,2,3, Ralph Patrick2,4,5, Joshua W. K. Ho2,4,6* and Michael D. O’Connor1,7* From 29th International Conference on Genome Informatics Yunnan, China. 3-5 December 2018 Abstract Background: Signaling pathways are the key biological mechanisms that transduce extracellular signals to affect transcription factor mediated gene regulation within cells. A number of computational methods have been developed to identify the topological structure of a specific signaling pathway using protein-protein interaction data, but they are not designed for identifying active signaling pathways in an unbiased manner. On the other hand, there are statistical methods based on gene sets or pathway data that can prioritize likely active signaling pathways, but they do not make full use of active pathway structure that link receptor, kinases and downstream transcription factors. Results: Here, we present a method to simultaneously predict the set of active signaling pathways, together with their pathway structure, by integrating protein-protein interaction network and gene expression data. We evaluated the capacity for our method to predict active signaling pathways for dental epithelial cells, ocular lens epithelial cells, human pluripotent stem cell-derived lens epithelial cells, and lens fiber cells. This analysis showed our approach could identify all the known active pathways that are associated with tooth formation and lens development. Conclusions: The results suggest that SPAGI can be a useful approach to identify the potential active signaling pathways given a gene expression profile.
    [Show full text]
  • From Pathway to Population–A Multiscale Model of Juxtacrine
    BMC Systems Biology BioMed Central Research article Open Access From pathway to population – a multiscale model of juxtacrine EGFR-MAPK signalling DC Walker*1, NT Georgopoulos2 and J Southgate2 Address: 1Department of Computer Science, University of Sheffield, Kroto Research Institute, North Campus, Broad Lane, Sheffield, S3 7HQ, UK and 2Jack Birch Unit of Molecular Carcinogenesis, Department of Biology, University of York, York YO10 5YW, UK Email: DC Walker* - [email protected]; NT Georgopoulos - [email protected]; J Southgate - [email protected] * Corresponding author Published: 26 November 2008 Received: 3 June 2008 Accepted: 26 November 2008 BMC Systems Biology 2008, 2:102 doi:10.1186/1752-0509-2-102 This article is available from: http://www.biomedcentral.com/1752-0509/2/102 © 2008 Walker et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Most mathematical models of biochemical pathways consider either signalling events that take place within a single cell in isolation, or an 'average' cell which is considered to be representative of a cell population. Likewise, experimental measurements are often averaged over populations consisting of hundreds of thousands of cells. This approach ignores the fact that even within a genetically-homogeneous population, local conditions may influence cell signalling and result in phenotypic heterogeneity. We have developed a multi-scale computational model that accounts for emergent heterogeneity arising from the influences of intercellular signalling on individual cells within a population.
    [Show full text]
  • The Study of Epidermal Growth Factor Receptor, 99Mtechnetium
    The Study of Epidermal Growth Factor Receptor, 99m Technetium-depreotide, and Tumour Markers in the Management of Neuroendocrine Tumours by Tahir H Shah A thesis submitted to University College London for the degree of Doctor of Medicine (Res) January 2009 Neuroendocrine Tumour Unit & Department of Oncology Royal Free and University College Medical School & University College London 91 Riding House Street, London W1W 7B I, Tahir H Shah, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. ABSTRACT Purpose: Neuroendocrine Tumours (NETs) are rare and therefore poorly understood. Their slow progression and general poor response to standard chemotherapy regimes implies that their tumour biology is significantly different from most common cancers. There have been major advances in the past few decades in the diagnosis and management of these tumours which are perceived to be associated with improvements in quality and length of life. However, since most NETs have metastasised extensively by the time of diagnosis it is a major challenge indeed to try and effect tumour regression – the ultimate goal of any anti-cancer therapy. Chapters: 2 & 3: The epidermal growth factor receptor (EGFR) is commonly expressed in human tumours and provides an important target for therapy. Several classes of agents including small molecule inhibitors and antibodies are currently under clinical evaluation. These agents have shown interaction with chemotherapeutic agents in vitro and in vivo . However, the mechanisms of these interactions are not clearly understood, particularly in regards to NETs. The purpose of this study was to investigate the expression of EGFR in NET tissue and to determine its mechanisms of action, as well as the effects of modulation of EGFR activation.
    [Show full text]
  • Hippo and Sonic Hedgehog Signalling Pathway Modulation of Human Urothelial Tissue Homeostasis
    Hippo and Sonic Hedgehog signalling pathway modulation of human urothelial tissue homeostasis Thomas Crighton PhD University of York Department of Biology November 2020 Abstract The urinary tract is lined by a barrier-forming, mitotically-quiescent urothelium, which retains the ability to regenerate following injury. Regulation of tissue homeostasis by Hippo and Sonic Hedgehog signalling has previously been implicated in various mammalian epithelia, but limited evidence exists as to their role in adult human urothelial physiology. Focussing on the Hippo pathway, the aims of this thesis were to characterise expression of said pathways in urothelium, determine what role the pathways have in regulating urothelial phenotype, and investigate whether the pathways are implicated in muscle-invasive bladder cancer (MIBC). These aims were assessed using a cell culture paradigm of Normal Human Urothelial (NHU) cells that can be manipulated in vitro to represent different differentiated phenotypes, alongside MIBC cell lines and The Cancer Genome Atlas resource. Transcriptomic analysis of NHU cells identified a significant induction of VGLL1, a poorly understood regulator of Hippo signalling, in differentiated cells. Activation of upstream transcription factors PPARγ and GATA3 and/or blockade of active EGFR/RAS/RAF/MEK/ERK signalling were identified as mechanisms which induce VGLL1 expression in NHU cells. Ectopic overexpression of VGLL1 in undifferentiated NHU cells and MIBC cell line T24 resulted in significantly reduced proliferation. Conversely, knockdown of VGLL1 in differentiated NHU cells significantly reduced barrier tightness in an unwounded state, while inhibiting regeneration and increasing cell cycle activation in scratch-wounded cultures. A signalling pathway previously observed to be inhibited by VGLL1 function, YAP/TAZ, was unaffected by VGLL1 manipulation.
    [Show full text]
  • Kremen Is Beyond a Subsidiary Co-Receptor of Wnt Signaling: an in Silico Validation
    Turkish Journal of Biology Turk J Biol (2015) 39: 501-510 http://journals.tubitak.gov.tr/biology/ © TÜBİTAK Research Article doi:10.3906/biy-1409-1 Kremen is beyond a subsidiary co-receptor of Wnt signaling: an in silico validation 1 2, 3 Hemn MOHAMMADPOUR , Saeed KHALILI *, Zahra Sadat HASHEMI 1 Department of Medical Immunology, Faculty of Medical Science, Tarbiat Modares University, Tehran, Iran 2 Department of Medical Biotechnology, Faculty of Medical Science, Tarbiat Modares University, Tehran, Iran 3 Department of Medical Biotechnology, School of Advanced Medical Technologies, Tehran University of Medical Science, Tehran, Iran Received: 01.09.2014 Accepted/Published Online: 12.02.2015 Printed: 15.06.2015 Abstract: Kremen-1 is a co-receptor of Wnt signaling, interacting with the Dkk-3 protein, which is an antagonist of the Wnt/β catenin pathway. In the present study we attempted to shed some light on the possible orientations of Kremen/Dkk-3 interactions, explaining the mechanisms of Kremen and Dkk-3 functions. Employing state-of-the-art software, a Kremen model was built and subsequently refined. The quality of the final model was evaluated using RMSD calculations. Ultimately, we used the Kremen model for docking analysis between Kremen and Dkk-3 molecules. A model built by the Robetta server showed the best quality scores. Near native coordination of the final model was verified getting < 2 Å RMSD values between our model and an experimentally resolved structure. Docking analysis indicates that one low energy orientation for Kremen/Dkk-3 involves all extracellular domains of Kremen, while another only involves the CUB and WSC domains.
    [Show full text]