Cytotoxic Drug Resistance Mechanisms PDF
Preview Cytotoxic Drug Resistance Mechanisms
Cytotoxic Drug Resistance Mechanisms M E T H O D S I N M O L E C U L A R M E D I C I N ETM John M. Walker, SERIES EDITOR 29. DNA Vaccines, edited by Douglas B. Lowrie and Robert Whalen, 1999 28. Cytotoxic Drug Resistance Mechanisms, edited by Robert Brown and Uta Böger-Brown, 1999 27. Clinical Applications of Capillary Electrophoresis, edited by Stephen M. Palfrey, 1999 26. Quantitative PCR Protocols, edited by Bernd Kochanowski and and Udo Reischl, 1999 25. Drug Targeting, edited by G. E. Francis and Cristina Delgado, 1999 24. Antiviral Chemotherapy Protocols, edited by Derek Kinchington and Raymond F. Schinazi, 1998 23. Peptidomimetics Protocols, edited by Wieslaw M. Kazmierski, 1998 22. Neurodegeneration Methods and Protocols, edited by Jean Harry and Hugh A. Tilson, 1998 21. Adenovirus Methods and Protocols, edited by William S. M. Wold, 1998 20. Sexually Transmitted Diseases Protocols, edited by Rosanna Peeling and P. Frederick Sparling, 1998 19. 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Gene Therapy Protocols, edited by Paul D. Robbins, 1997 6. Molecular Diagnosis of Cancer, edited by Finbarr E. Cotter, 1996 5. Molecular Diagnosis of Genetic Diseases, edited by Rob Elles, 1996 4. Vaccine Protocols, edited by Andrew Robinson, Graham H. Farrar, and Christopher N. Wiblin, 1996 3. Prion Diseases, edited by Harry F. Baker and Rosalind M. Ridley, 1996 2. Human Cell Culture Protocols, edited by Gareth E. Jones, 1996 1. Antisense Therapeutics, edited by Sudhir Agrawal, 1996 M E T H O D S I N M O L E C U L A R M E D I C I N ETM Cytotoxic Drug Resistance Mechanisms Edited by Robert Brown University of Glasgow, Glasgow, UK and Uta Böger-Brown BioMedEx, Killearn, UK Humana Press Totowa, New Jersey © 1999 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular Medicine™is a trademark of The Humana Press Inc. All authored papers, comments, opinions, conclusions, or recommendations are those of the author(s), and do not necessarily reflect the views of the publisher. This publication is printed on acid-free paper. ∞ ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials. Cover illustration: Fig. 1 from Chapter 17, “Analysis of the p53 Status of Tumors: An Overview of Methods,” by Jonas Bergh. Cover design by Patricia F. Cleary. 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Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1 Library of Congress Cataloging in Publication Data Main entry under title: Methods in molecular medicine™. Cytotoxic Drug Resistance Mechanisms / edited by Robert Brown and Uta Böger-Brown. p. cm. — (Methods in molecular medicine™ ; 28) Includes index. ISBN 0-89603-603-0 (alk. paper) 1. Drug resistance in cancer cells—Research—Methodology. I. Brown, Robert , 1956 Jan. 8– II. Böger-Brown, Uta. III. Series. [DNLM: 1. Drug Resistance, Neoplasm. 2. Antineoplastic Agents—Pharmacology. 3. DNA Damage. 4. Genetic Techniques. QZ 267 C9975 1999] RC271.C5C97 1999 616.99'4061—dc21 DNLM/DLC for Library of Congress 98-43528 CIP Preface There is now a range of cytotoxic drugs that have considerable clinical usefulness in producing responses in tumors and even, in a small proportion of cases, cure. However, the acquisition of drug resistance is a major clinical problem and is perhaps the main limiting factor in successful treatment of cancer. Thus, a tumor initially sensitive to chemotherapy will, in the majority of cases, eventually recur as a resistant tumor, which will then progress. Much of our understanding of drug resistance mechanisms comes from the study of tumor cell lines grown in tissue culture. We now understand many of the mo- lecular mechanisms that can lead to a cell acquiring resistance to antican- cer drugs; however, we still do not know which mechanism(s) are those most relevant to the problem of clinical drug resistance. Indeed, given that many of the cytotoxic anticancer drugs were discovered by random screening, it is un- clear what features give a clinically useful anticancer drug a sufficient thera- peutic index to be of value. The aim of Cytotoxic Drug Resistance Mechanismsis to provide proto- cols that are appropriate for examining the mechanisms of cellular resistance to anticancer cytotoxics in human tumor samples. Tumor cell lines have been enormously useful as experimental models of drug resistance mechanisms, however they have limitations and we need to address the relevance of such mechanisms in patients’ tumors. Examining drug resistance in tumors is much more problematic than in cell lines. Not least, because it is difficult to define the cellular sensitivity or resistance of a tumor sample. A number of assays of in vitro sensitivity have been developed to address this question. Since the realization that many anticancer cytotoxics kill cells by an apoptotic process, these cell sensitivity assays have come to include in vitro and in vivo mea- sures of apoptosis. To an outside observer, drug resistance is often taken as being synony- mous with the Multi-Drug Resistance Phenotype, conferred by MDR1/P-gly- coprotein and related genes. This has been an intensively studied mechanism of resistance and is of clear clinical relevance. A large number of studies in this field have now led to a consensus on how MDR1/Pgp should be measured in tumor samples; this consensus should be a role model for future studies on other resistance mechanisms. Though P-glycoprotein-mediated drug efflux is important, it is clearly not the only mechanism of relevance to clinical drug v vi Preface resistance. A number of studies would suggest that MDR1/Pgp is not the rea- son for the acquired drug resistance of many solid tumors. There is a large body of circumstantial evidence for most of the cytotoxics described in Cytotoxic Drug Resistance Mechanisms, all of which supports DNA as the crucial target. Delivering sufficient drug to the chemotherapeutic target is obviously important. Although the methods presented here do not deal with pharmacological resistance, means of measuring the amount of DNA damage induced by various cytotoxics are described. Undoubtedly the most exciting emerging area in drug resistance during the last five years has been the role of drug-induced apoptosis. A number of genes have now been impli- cated in modulating the ability of a cell to undergo drug-induced apoptosis, including DNA repair proteins, p53-dependent pathways, and the bcl2 gene family. Such mechanisms modulate resistance in a manner that will be en- tirely independent of the amount of drug delivered. The relevance of these apoptosis-modulating mechanisms to clinical resistance will be an important research area over the next years. Drug resistance can be clinically defined as a tumor's lack of response to therapy. It is possible that there are yet unknown mechanisms of clinical resis- tance still to be identified in experimental models. Therefore examining ge- netic alterations in clinical samples without prior assumption may prove invaluable. Molecular cytogenetics may prove to be one such approach that will provide important information. For each of the methods described in Cytotoxic Drug Resistance Mecha- nisms, the most important ingredient is the effective admixture of clinical and laboratory scientists that allows correlations between molecular and biochemi- cal data and the clinical responses of patients to be identified. With that in mind, I would like to thank all the surgeons, pathologists, biologists, medical oncologists, and other colleagues who have contributed their correct ingredi- ents to the elucidation of drug resistance phenomena. Robert Brown and Uta Böger-Brown Contents Preface .............................................................................................................v Contributors.....................................................................................................ix 1 Drug Resistance: The Clinical Perspective D. Alan Anthoney and Stanley B. Kaye ............................................1 2 Cell Sensitivity Assays: Clonogenic Assay Jane A. Plumb .....................................................................................17 3 Cell Sensitivity Assays: The MTT Assay Jane A. Plumb ....................................................................................25 4 Cell Sensitivity Assays: Detection of Apoptotic Cells In Vitro Using the TUNEL Assay Neil A. Jones and Caroline Dive .......................................................31 5 Analysis of Apoptosis in Tissue Sections Vicki Save, Philip J. Coates, and Peter A. Hall ...............................39 6 Measurement of P-glycoprotein Function Henk J. Broxterman ...........................................................................53 7 MeasuringMDR-1 by Quantitative RT-PCR Susan E. Bates, Zhirong Zhan, Joanna Regis, and Erick Gamelin ..........................................................................63 8 Microtiter Plate Technique for the Measurement of Glutathione in Fresh and Cryopreserved Lymphoblasts Using the Enzyme Recycling Method Pamela R. Kearns and Andrew G. Hall ............................................83 9 Measurement of Reduced Glutathione Using High-Pressure Liquid Chromatography Linda A. Hogarth, Celia M. A. Rabello, and Andrew G. Hall..........91 10 Topoisomerase I and II Activity Assays Philippe Pourquier, Glenda Kohlhagen, Li-Ming Ueng, and Yves Pommier ........................................................................95 11 5-Fluorouracil Metabolizing Enzymes Howard L. McLeod, Lesley H. Milne, and Stephen J. Johnston ............................................................111 vii viii Contents 12 Measuring DNA Adducts by Immunoassay (ELISA) Michael J. Tilby .................................................................................121 13 Measuring Drug-DNA Adducts in Individual Cells Adrian J. Frank..................................................................................129 14 Measurement of Drug-Induced DNA Interstrand Crosslinking Using the Single-Cell Electrophoresis (Comet) Assay Victoria J. Spanswick, Janet M. Hartley, Timothy H. Ward, and John A. Hartley..........................................................................143 15 PCR Analysis of Microsatellite Instability Gillian L. Hirst ...................................................................................155 16 O6-Alkylguanine-DNA Alkyltransferase Assay Amanda J. Watson and Geoffrey P. Margison .............................167 17 Analysis of the p53 Status of Tumors: An Overview of Methods Jonas Bergh ......................................................................................179 18 Bcl-2 Family Immunohistochemistry Lloyd R. Kelland and Philip J. Beale ..............................................201 19 Genetic Analysis of Drug Resistance by Fluorescence In Situ Hybridization W. Nicol Keith ...................................................................................209 20 Genetic Analysis of Drug Resistance by Reverse In Situ Hybridization W. Nicol Keith....................................................................................225 Index............................................................................................................235 Contributors D. ALAN ANTHONEY • CRC Department of Medical Oncology, Beatson Laboratories, University of Glasgow, Glasgow, UK PHILIP J. BEALE • CRC Centre for Cancer Therapeutics, The Institute of Cancer Research, Sutton, Surrey, UK SUSAN E. BATES • Medicine Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD JONAS BERGH • Department of Oncology, University Hospital, Uppsala, Sweden HENK J. BROXTERMAN • Department of Medical Oncology, Free University Hospital, Amsterdam, The Netherlands PHILIP J. COATES• Department of Molecular and Cellular Pathology, University of Dundee, Dundee, UK CAROLINE DIVE• Molecular and Cellular Pharmacology Group, University of Manchester, School of Biological Sciences, Manchester, UK ADRIAN J. FRANK• Leukaemia Research Fund Laboratory, University of Newcastle Upon Tyne, Medical School, Newcastle Upon Tyne, UK ERICK GAMELI • Medicine Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD ANDREW G. HALL •Paediatric Oncology Laboratory, Cancer Research Unit, University of Newcastle upon Tyne, Medical School, Newcastle upon Tyne, UK PETER A. HALL • Department of Molecular and Cellular Pathology, University of Dundee, Dundee, UK JANET M. HARTLEY • CRC Drug-DNA Interactions Research Group, Department of Oncology, University College London Medical School, London, UK JOHN A. HARTLEY • CRC Drug-DNA Interactions Research Group, Department of Oncology, University College London Medical School, London, UK GILLIAN L. HIRST • CRC Department of Medical Oncology, Beatson Laboratories, University of Glasgow, Glasgow, UK LINDA A. HOGARTH• Paediatric Oncology Laboratory, Cancer Research Unit, University of Newcastle Upon Tyne, Medical School, Newcastle Upon Tyne, UK ix x Contributors STEPHEN J. JOHNSTON • Department of Medicine and Therapeutics, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, UK NEIL A. JONES• Molecular and Cellular Pharmacology Group, University of Manchester, School of Biological Sciences, Manchester, UK STANLEY B. KAYE • CRC Department of Medical Oncology, Beatson Laboratories, University of Glasgow, Glasgow, UK PAMELA R. KEARNS • Paediatric Oncology Laboratory, Cancer Research Unit, University of Newcastle Upon Tyne, Medical School, Newcastle Upon Tyne, UK W. NICOL KEITH • CRC Department of Medical Oncology, Beatson Laboratories, University of Glasgow, Glasgow, UK LLOYD R. KELLAND • CRC Centre for Cancer Therapeutics, The Institute of Cancer Research, Sutton, Surrey, UK GLENDA KOHLHAGEN • Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD GEOFFREY P. MARGISON • Department of Carcinogenesis, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK HOWARD L. MCLEOD • Department of Medicine and Therapeutics, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, UK LESLEY H. MILNE • CRC Department of Medical Oncology, Beatson Laboratories, University of Glasgow, Glasgow, UK JANE A PLUMB • CRC Department of Medical Oncology, Beatson Laboratories, University of Glasgow, Glasgow, UK YVES POMMIER • Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD PHILIPPE POURQUIER• Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD VICKI SAVE • Department of Molecular and Cellular Pathology, University of Dundee, Dundee, UK CELIA M. A. RABELLO • Paediatric Oncology Laboratory, Cancer Research Unit, University of Newcastle Upon Tyne, Medical School, Newcastle Upon Tyne, UK JOANNA REGIS • Medicine Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD VICTORIA J. SPANSWICK • CRC Drug-DNA Interactions Research Group, Department of Oncology, University College London Medical School, London, UK
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