Molecular Biology in Cellular Pathology Molecular Biology in Cellular Pathology. Edited by John Crocker and Paul G. Murray  2003 John Wiley & Sons, Ltd ISBN: 0-470-84475-2 Molecular Biology in Cellular Pathology Edited by John Crocker Department of Cellular Pathology, Heartlands Hospital, Birmingham, UK Paul G. Murray Department of Pathology, The Medical School University of Birmingham, Birmingham, UK Copyright  2003 John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex PO19 8SQ, England Telephone (+44) 1243 779777 Email (for orders and customer service enquiries): cs-books@wiley.co.uk Visit our Home Page on www.wileyeurope.com or www.wiley.com All Rights Reserved. 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Includes bibliographical references and index. ISBN 0-470-84475-2 (paper : alk. paper) 1. Pathology, Molecular. 2. Pathology, Cellular. [DNLM: 1. Genetic Techniques. 2. Cell Physiology. 3. Cells – pathology. QZ 52 M718 20003] I. Crocker, J. II. Murray, Paul, Ph. D. III. Molecular biology in cellular p athology. RB43.7 .M6336 2003 611  .018–dc21 2002154112 British Library Cataloguing in Publication D ata A catalogue record for this book is av ailable from the British Library ISBN 0-470-84475-2 Typeset in 10.5/13pt Times by Laserwords Private Limited, Chennai, India Printed and bound in Great Britain by TJ International, Padstow, Cornwall This book is printed on acid-free paper responsibly manufactured from sustainable forestry in which at least two trees are planted for each one used for paper production. With love to our families Contents Preface xiii Preface to Molecular Biology in Histopathology xv List of Contributors xvii 1 Blotting Techniques: Methodology and Applications 1 Fiona Watson and C. Simon Herrington 1.1 Introduction 1 1.2 Blotting techniques 1 1.3 References 15 2 In-situ Hybridisation in Histopathology 19 Gerald Niedobitek and Hermann Herbst 2.1 Introduction 19 2.2 Experimental conditions 20 2.3 Probes and labels 23 2.4 Controls and pitfalls 27 2.5 Double-labelling 29 2.6 Increasing the sensitivity of ISH 31 2.7 What we do in our laboratories 33 2.8 Applications of ISH: examples 35 2.9 Perspective 39 2.10 References 40 3 DNA Flow Cytometry 49 M.G. Ormerod 3.1 Introduction 49 3.2 Definitions and terms 49 3.3 Dye used for DNA analysis 50 3.4 Sample preparation for DNA analysis 52 viii CONTENTS 3.5 Analysis of the DNA histogram 53 3.6 Quality control 53 3.7 Computer analysis of the DNA histogram 55 3.8 Multiparametric measurement 57 3.9 Acknowledgements 59 3.10 References 59 4 Interphase Cytogenetics 61 Sara A. Dyer and Jonathan J. Waters 4.1 Introduction 61 4.2 Interphase cytogenetics 62 4.3 Applications 67 4.4 Conclusion 76 4.5 References 77 5 Oncogenes 79 Fiona Macdonald 5.1 Introduction 79 5.2 Identification of the oncogenes 79 5.3 Functions of the proto-oncogenes 80 5.4 Mechanism of oncogene activation 89 5.5 Oncogenes in colorectal cancer 91 5.6 Oncogenes in breast cancer 94 5.7 Oncogenes in lung cancer 95 5.8 Oncogenes in haematological malignancies 96 5.9 Other cancers 99 5.10 Conclusion 100 5.11 References 100 6 Molecular and Immunological Aspects of Cell Proliferation 105 Karl Baumforth and John Crocker 6.1 The cell cycle and its importance in clinical pathology 105 6.2 Molecular control of the cell cycle 108 6.3 Cell cycle control 111 6.4 The cell cycle and cancer 112 6.5 Immunocytochemical markers of proliferating cells 115 6.6 References 133 6.7 Further Reading 135 7 Interphase Nucleolar Organiser Regions in Tumour Pathology 137 Massimo Derenzini, Davide Trer´e, Marie-Fran¸coise O’Donohue and Dominique Ploton CONTENTS ix 7.1 Introduction 137 7.2 The AgNORs 138 7.3 NOR silver-staining 142 7.4 Quantitative AgNOR analysis 145 7.5 AgNORs as a parameter of the level of cell proliferation 146 7.6 Application of the AgNOR technique to tumour pathology 147 7.7 What future for AgNORs in tumour pathology? 151 7.8 References 152 8 Apoptosis and Cell Senescence 153 Lee B. Jordan and David J. Harrison 8.1 Introduction 153 8.2 Apoptosis 153 8.3 Cell senescence 174 8.4 Summary 178 8.5 References 179 9 The Polymerase Chain Reaction 193 Ti mothy Diss 9.1 Introduction 193 9.2 Principles 194 9.3 Analysis of products 197 9.4 RT-PCR 199 9.5 Quantitative PCR 200 9.6 DNA and RNA extraction 200 9.7 Correlation of the PCR with morphology 201 9.8 Problems 202 9.9 Applications 202 9.10 Diagnostic applications 203 9.11 Infectious diseases 209 9.12 Identity 209 9.13 The future 210 9.14 References 210 9.15 Online information 212 10 Laser Capture Microdissection: Techniques and Applications in the Molecular Analysis of the Cancer Cell 213 Amanda Dutton, Victor Lopes and Paul G. Murray 10.1 Introduction 213 10.2 The principle of LCM 214 10.3 Technical considerations 216 10.4 Advantages and disadvantages of LCM 217 x CONTENTS 10.5 Applications of LCM 222 10.6 Future perspectives 229 10.7 Acknowledgements 229 10.8 References 229 11 The In-situ Polymerase Chain Reaction 233 John J. O’Leary, Cara Martin and Orla Sheils 11.1 Introduction 233 11.2 Overview of the methodology 234 11.3 In-cell PCR technologies 235 11.4 In-cell amplification of DNA 238 11.5 Detection of amplicons 242 11.6 Reaction, tissue and detection controls for use with in-cell DNA PCR assays 243 11.7 In-cell RNA amplification 244 11.8 Problems encountered with in-cell PCR amplification 246 11.9 Amplicon diffusion and back diffusion 247 11.10 Future work with in-cell PCR-based assays 247 11.11 References 249 12 TaqMan  Technology and Real-Time Polymerase Chain Reaction 251 John J. O’Leary, Orla Sheils, Cara Martin, and Aoife Crowley 12.1 Introduction 251 12.2 Probe technologies 252 12.3 TaqMan  probe and chemistry (first generation) 254 12.4 Second generation TaqMan  probes 256 12.5 Hybridisation 258 12.6 TaqMan  PCR conditions 259 12.7 Standards for quantitative PCR 260 12.8 Interpretation of results 261 12.9 End-point detection 262 12.10 Real-time detection 263 12.11 Relative quantitation 263 12.12 Reference genes 264 12.13 Specific TaqMan  PCR applications 265 12.14 References 268 13 Gene Expression Analysis Using Microarrays 269 Sophie E. Wildsmith and Fiona J. Spence 13.1 Introduction 269 13.2 Microarray experiments 269 CONTENTS xi 13.3 Data analysis 273 13.4 Recent examples of microarray applications 284 13.5 Conclusions 284 13.6 Acknowledgements 284 13.7 References 284 13.8 Further Reading 286 13.9 Useful websites 286 14 Comparative Genomic Hybridisation in Pathology 287 Marjan M. Weiss, Mario A.J.A. Hermsen, Antoine Snijders, Horst Buerger, Werner Boecker, Ernst J. Kuipers, Paul J. van Diest andGerritA.Meijer 14.1 Introduction 287 14.2 Technique 289 14.3 Data analysis 292 14.4 Applications 293 14.5 Clinical applications 299 14.6 Screening for chromosomal abnormalities in fetal and neonatal genomes 299 14.7 Future perspectives 300 14.8 Acknowledgements 301 14.9 References 301 15 DNA Sequencing and the Human Genome Project 307 Philip Bennett 15.1 Introduction 307 15.2 DNA sequencing: the basics 308 15.3 Applications of DNA sequencing 318 15.4 The Human Genome Project 320 15.5 References 327 15.6 Further Reading 327 15.7 Useful websites 328 16 Monoclonal Antibodies: The Generation and Application of ‘Tools of the Trade’ Within Biomedical Science 329 Paul N. Nelson, S. Jane Astley and Philip Warren 16.1 Introduction 329 16.2 Antibodies and antigens 331 16.3 Polyclonal antibodies 332 16.4 Monoclonal antibody development 333 16.5 Monoclonal antibody variants 338 16.6 Monoclonal antibody applications 341 xii CONTENTS 16.7 Therapy 345 16.8 Specific applications 346 16.9 Conclusions 347 16.10 Acknowledgements 347 16.11 References 347 17 Proteomics 351 Kathryn Lilley, Azam Razzaq and Michael J. Deery 17.1 Introduction 351 17.2 Definitions and applications 352 17.3 Stages in proteome analysis 352 17.4 Future directions 368 17.5 References 368 Index 371 [...]... protein kinases using exogenous substrates, in: Current Protocols in Molecular Biology, Vol 4, 18.8.1–18.8.19 (eds FM Ausubel, R Brent, RE Kingston, DD Moore, JG Seidman, JA Smith and K Struhl) John Wiley & Sons, Inc ., New York Celis JE (1998) Cell Biology A Laboratory Handbook, Vol 4, pp 454–6 Academic Press, San Diego Celis JE, Kruhøffer M, Gromova I, Frederiksen C, Østergaard M, Thykjaer T, Gromov P,... RNA In Current protocols in molecular biology Vol 1, 4.0.3–4.2.9 (eds FM Ausubel, R Brent, RE Kingston, DD Moore, JG Seidman, JA Smith and K Struhl) John Wiley & Sons, Inc ., New York Kouklis PD, Hutton E and Fuchs E (1994) Making a connection: Direct binding between keratin intermediate filaments and desmosomal proteins J Cell Biol 127: 1049–60 Kramer MH, Hermans J, Wijburg E, Philippo K, Geelen E, van... Muenster, Germany John Crocker Department of Cellular Pathology, Birmingham Heartlands Hospital, Bordesley Green East, Birmingham B9 5SS, UK Aoife Crowley Department of Pathology, The Coombe Women’s Hospital Dublin and The Department of Histopathology, Trinity College Dublin, Ireland Michael J Deery Inpharmatica Ltd, 60 Charlotte Street, London W1T 2NU, UK Massimo Derenzini Universit` di Bologna, Dipartimento... Service, Birmingham Women’s Hospital, Edgbaston, Birmingham B15 2TG, UK David J Harrison Department of Pathology, University of Edinburgh, Edinburgh, UK Hermann Herbst Gerhard-Domagk-Institut f¨ r Pathologie, u Westf¨ lische Wilhems-Universit¨ t, a a Domagkstr 1 7, 48149 M¨ nster, Germany u Mario A.J.A Hermsen Department of Pathology, VU Medical Centre, Amsterdam, The Netherlands C Simon Herrington... Building O, Downing Site, Cambridge CB2 1QW, UK Victor Lopez Department of Pathology, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK Fiona MacDonald West Midlands Regional Genetics Laboratory, Birmingham Women’s Hospital NHS Trust, Edgbaston, Birmingham B15 2TG, UK Cara Martin Department of Pathology, The Coombe Women’s Hospital Dublin and The Department of Histopathology,... Pathologisches Institut, Friedrich-Alexander-Universit¨ t, a Krankenhausstr 8–1 0, 91054 Erlangen, Germany Marie-Fran¸ oise O’Donohue c CNRS UMR 614 2, Facult´ de M´ dicine, e e Reims Cedex, France John J O’Leary Department of Pathology, The Coombe Women’s Hospital Dublin and The Department of Histopathology, Trinity College Dublin, Ireland Michael G Ormerod 34 Wray Way, Reigate RH2 0DE, UK Dominique Ploton... of RNA binding proteins and their function are: (a) the correct folding and packaging of pre-rRNA mediated through the direct binding of nucleolin to two mutually exclusive RNA sequences (Ginisty et al ., 2001); (b) Hu proteins, which are RNA binding proteins postulated to regulate gene transcription at the post-transcriptional level (Chung et al ., 1996); (c) proteins involved in pre-mRNA splicing such... UMR 614 2, Facult´ de M´ dicine, e e Reims Cedex, France Azam Razzaq Cambridge Centre for Proteomics, University of Cambridge, Department of Biochemistry, Building O, Downing Site, Cambridge CB2 1QW, UK Orla Sheils Department of Pathology, The Coombe Women’s Hospital Dublin and The Department of Histopathology, Trinity College Dublin, Ireland Antoine Snijders UCSF Cancer Centre, San Francisco, USA Fiona... different combinations of RNA – binding domains determine the RNA binding specificity of nucleolin J Biol Chem 276: 14338–43 Grasser FA, Sauder C, Haiss P, Hille A, Konig S, Gottel S, Kremmer E, Leinenbach HP, Zeppezauer M and Mueller-Lantzsch N (1993) Immunological detection of proteins associated with the Epstein-Barr virus nuclear antigen 2A Virology 195: 550–60 Hall KB (2002) RNA – protein interactions... their use 1.2 Blotting techniques The Southern Blot This technique, which is used to detect specific sequences within mixtures of DNA, was first described by E.M Southern in 1975 (Southern, 1975) In a Molecular Biology in Cellular Pathology Edited by John Crocker and Paul G Murray  2003 John Wiley & Sons, Ltd ISBN: 0-4 7 0-8 447 5-2 2 BLOTTING TECHNIQUES: METHODOLOGY AND APPLICATIONS Intact RNA or digested . Molecular Biology in Cellular Pathology Molecular Biology in Cellular Pathology. Edited by John Crocker and Paul G. Murray  2003 John Wiley & Sons, Ltd ISBN: 0-4 7 0-8 447 5-2 Molecular Biology. appears in print may not be available in electronic books. Library of Congress Cataloging -in- Publication Data Molecular biology in cellular pathology / edited by John Crocker, Paul G. Murray. . Molecular biology in histopathology, c1994. Includes bibliographical references and index. ISBN 0-4 7 0-8 447 5-2 (paper : alk. paper) 1. Pathology, Molecular. 2. Pathology, Cellular. [DNLM: 1. Genetic