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Differential Metallothionein Isoform Expression As Biomarkers of Cadmium Exposure in Human Urothelium

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Differential Metallothionein Isoform Expression As Biomarkers of Cadmium Exposure in Human Urothelium Differential Metallothionein Isoform Expression as Biomarkers of Cadmium Exposure in Human Urothelium Rhiannon McNeill Doctor of Philosophy University of York Biology June 2017 Abstract Abstract The metallothionein (MT) superfamily consists of 11 genes whose proteins bind to and sequester metals within cells. Although the presence of so many isoforms raises the possibility of differing metal specificities and functions, they have rarely been discriminated in the literature. This thesis investigated the expression and inducibility of MT isoforms in normal human urothelium with the aim of determining whether specific MT isoforms represent discriminatory biomarkers of cadmium exposure, which has been increasingly linked to the development of urothelial cancer (UC) in recent years. Specialised techniques already established were used for the cell, tissue and organ culture of normal human urothelium. These cultures were exposed to cadmium concentrations reflective of in vivo human exposure and analysed using isoform-specific primers and antibodies. The results revealed the extent of differential expression of the MT isoforms, including preferential metal activation; for example, zinc could highly induce MT-1G transcript expression but could only cause minimal transcript induction of MT-1M. This demonstrates the importance of distinguishing between all known isoforms when determining MT expression. The discriminatory approach used in this thesis allowed the identification of two MT-1 isoforms, MT-1A and MT-1M, whose protein induction was highly specific to cadmium exposure. Although isoform transcript induction was observed to be transient, the protein expression of MT-1A persisted for at least 6 weeks post exposure, consistent with a metal sequestration role. Investigation using spectroscopic techniques additionally suggested that cadmium could penetrate the protective urothelial barrier and enter the underlying urothelial cells, where it may be sequestered by MT within these long-lived cells, thus serving as a long-term source for chronic exposure. Overall the results suggest that MT-1 isoforms may be useful as urothelial biomarkers of cadmium exposure, potentially allowing the identification of individuals ‘at risk’ of developing UC and additionally, stratifying a subset of cadmium- induced UC. 2 Abbreviations Abbreviations ABS – Adult Bovine Serum ARE – Antioxidant Response Element AUM – Asymmetric Unit Membrane BASQ – Basal-squamous-like BLAST – Basic Logical Alignment and Search Tool bp – Base Pair(s) BPE – Bovine Pituitary Extract BSA – Bovine Serum Albumin Ca2+ - Calcium Ion CdCl2 – Cadmium Chloride cDNA – Complementary Deoxyribonucleic Acid CK – Cytokeratin CLDN - Claudin CO2 – Carbon Dioxide CT – Cholera Toxin DAB - Diaminobenzidine DEPC – Diethyl Pyrocarbonate dH2O – DEPC-treated Water DMEM – Dulbecco’s Modified Eagle’s Serum DMSO – Dimethyl Sulphoxide DNA – Deoxyribonucleic Acid DNMT – DNA Methyltransferase dNTP – Deoxynucleotide Triphosphate DTT – Dithiothrietol EDTA- Ethylenediaminetetra-acetic Acid EGF – Epidermal Growth Factor 3 Abbreviations EGFR – Epidermal Growth Factor Receptor FBS – Foetal Bovine Serum GAPDH - Glyceraldehyde 3-phoshate Dehydrogenase GRE – Glucocorticoid Response Element H2O - Water H&E – Haemotoxylin and Eosin HIF – Hypoxia Inducible Factor HRP – Horseradish Peroxidase IL – Interleukin ISUP – Internation Society of Urological Pathology kb – Kilobase kDa – Kilodalton KSFM – Keratinocyte Serum-Free Medium KSFMc – Keratinocyte Serum-Free Medium (Complete) M – Molar MAPK – Mitogen-Activated Protein Kinase MBP – Methyl-Binding Proteins MES - Morpholino-ethanesulfonic acid mg – Milligrams MI – Muscle Invasive mL – Millilitres mM – Millimolars MOPS - Morpholino-propanesulfonic acid mRNA – Messenger Ribonucleic Acid MT – Metallothionein N2 – Nitrogen NAC – N-acetyltransferase 4 Abbreviations ng – Nanograms NGC – Non-Genotoxic Carcinogen NHU – Normal Human Urothelial nM – Nanomolar nm – Nanometres NMI – Non-mucle invasive O2 – Oxygen °C – Degrees Centigrade P# – Passage Number PBS – Phosphate Buffered Saline PCR – Polymerase Chain Reaction PI – Proteinase Inhibitor PPAR – Peroxisome Proliferator-Activated Receptor PVDF - Polyvinylidine Fluoride PWM – Positional Weight Matrices ROS – Reactive Oxygen Species RNA – Ribonucleic Acid rpm – Rotations Per Minute RPMI - Roswell Park Memorial Institute RT – Reverse Transcriptase SDS – Sodium Dodecyl Sulphate SDS – PAGE – SDS – polyacrylamide gel electrophoresis SF - Sulforaphane TBE – Tris-Borate-EDTA TBS – Tris Buffered Saline TER – Transepithelial Electrical Resistance TF – Transcription Factor 5 Abbreviations TFBS – Transcription Factor Binding Sites TI – Trypsin Inhibitor TJ – Tight Junction Tm – Melting Temperature TNM – Tumour-Node-Metastasis TURBT – Transurethral Resection of Bladder Tumour TV – Trypsin Versene UPK – Uroplakin v/v – Volume/Volume WHO – World Health Organisation w/v – Weight/Volume ZnT – Zinc Transporter ZO – Zona Occludens µg – Micrograms µL – Microlitres µM - Micromolar µm - Micrometres 6 List of Contents List of Contents Abstract ............................................................................................................................. 2 Abbreviations .................................................................................................................... 3 List of Contents ................................................................................................................. 7 List of Tables .................................................................................................................. 15 List of Figures ................................................................................................................. 16 Acknowledgements ......................................................................................................... 21 Declaration ...................................................................................................................... 22 Chapter 1: Introduction ................................................................................................... 23 1.1 The Urothelium ..................................................................................................... 23 1.1.2 Structure and Organisation ............................................................................ 23 1.1.3 Barrier Function ............................................................................................. 25 1.1.4 In Vitro Models of Human Urothelium.......................................................... 26 1.1.5 Organ Culture ................................................................................................. 27 1.2 Urothelial Cancer .................................................................................................. 28 1.2.1 Grading and Staging of Tumours ................................................................... 28 1.2.2 Classification of Urothelial Cancer ................................................................ 29 1.2.3 Epidemiology and Statistics ........................................................................... 34 1.2.4 Causes of Urothelial Carcinogenesis ............................................................. 34 1.2.5 Cellular Origin of Urothelial Carcinoma ....................................................... 35 1.2.6 Clinical Management of Urothelial Cancer ................................................... 36 1.3 Heavy Metal Exposure .......................................................................................... 38 1.3.1 Examples and Sources of Heavy Metals ........................................................ 38 1.3.2 Exposure and Correlation with Carcinogenesis ............................................. 38 1.3.3 General Mechanisms of Heavy Metal-Carcinogenesis .................................. 39 1.4 Cadmium Exposure ............................................................................................... 41 1.4.1 Sources of Exposure....................................................................................... 41 7 List of Contents 1.4.2 Types of Exposure ......................................................................................... 42 1.4.3 Implication in Urothelial Carcinogenesis ...................................................... 43 1.4.4 Mechanisms of Cadmium-Induced Carcinogenesis ....................................... 44 1.5 The Metallothionein Proteins ................................................................................ 49 1.5.1 3-Dimensional (3-D) Structure and Conformation ........................................ 49 1.5.2 MT Gene Superfamily ................................................................................... 50 1.5.3 MT Gene Transcriptional Regulation ............................................................ 52 1.5.4 Proposed Functions of MT ............................................................................. 53 1.5.5 Metallothionein Isoforms ............................................................................... 58 1.5.6 MT Expression in Cadmium-Induced Urothelial Carcinogenesis ................. 61 1.5.7 MT Expression in Cadmium-Induced Urothelial Carcinogenesis – Cause or Consequence? ......................................................................................................... 64 1.6 Thesis Aims and Objectives. ................................................................................. 68 1.6.1 Aims ..............................................................................................................
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