Cytotoxic Drug Resistance Mechanisms (Methods in Molecular Medicine)

252 Pages·1999·1.065 MB·English

by  Robert Brown, Uta Boger-Brown

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
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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.
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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