|PR2017-00737
`
`Genentech 2137
`Genentech 2137 '
`Hospira v. Genentech
`Hospira v. Genentech
`IPR2017-00737
`
`

`

`The Basic Science of
`Oncology
`
`Second Edition
`
`Editors
`
`Ian F. Tannock, M.D., Ph.D.
`Richard P. Hill, Ph.D.
`
`The Ontario Cancer Institute
`and
`The University of Toronto
`Toronto, Ontario
`Canada
`
`McGraw-Hill, lnc.
`Health Professions Division
`
`San Francisco Auckland
`St. Louis
`New York
`Bogota Caracas
`Lisbon
`London Madrid
`Mexico Milan Montreal New Delhi
`Paris
`
`San Juan
`
`Singapore
`
`Sydney
`
`Tokyo
`
`Toronto
`
`

`

`
`
`THE BASIC SCIENCE OF ONCOLOGY
`
`Copyright © 1992 by McGraw-Hill, Inc. All rights reserved.
`Printed in the United States of America. Except as permitted un—
`der the United States Copyright Act of 1976, no part of this pub-
`lication may be reproduced or distributed in any form or by any
`means, or stored in a data base or retrieval system, without the
`prior written permission of the publisher.
`
`1234567890MALMAL98765432
`
`This book was set in Baskerville by Beljan, Ltd.
`The editors were jane E. Pennington and Lester A. Sheinis.
`The production supervisor was Roger Kasunic.
`The cover was designed by Carol Matsuyama.
`Malloy Lithographing, Inc., was printer and binder.
`
`ISBN 0-07-105407-3
`
`Library of Congress Cataloging-in-Publication Data
`
`The Basic science of oncology / edited by Ian F. Tannock, Richard
`P. Hill.
`p. cm.
`Includes bibliographical references and index.
`ISBN 0-07-105407-3
`1. Cancer.
`2. Oncology.
`Richard P.
`
`1. Tannock, Ian F.
`
`II. Hill,
`
`[DNLM: 1. Neoplasms. QZ 200 B3115]
`RC261.B37 1992
`616.99’ 4—dc20
`DNLM/DLC
`
`for Library of Congress
`
`92-2884CIP
`
`

`

`Contents
`AbouttheEditorsandContributors..................................................vii
`
`Preface ............................................................................ ix
`
`1. Introduction: CancerasaCellularDisease...............1
`
`Richard P. Hill and Ian F. Tarmac/c
`
`PART 1: CANCER CAUSATION
`
`2. EpidemiologyofCancer...............7
`Norman F. Boyd
`
`3. MethodsofGeneticAnalysis................................................
`Jeremy Squire and RobertA. Phillips
`
`...23
`
`4-. GeneticBasisofCancer. 4-1
`
`JeremySquireandRoberlA. Phillips
`
`SOncogenes.
`.. 61
`MarkD. Mina’eri and Anthony J Pawson
`
`6. Viruses and Cancer. .
`Sam Benchimol
`
`.
`
`.
`
`. ...................................................... 88
`
`7. Chemical Carcinogenesis......................................................102
`Michael C. Archer
`
`8/.RadiationCarcinogenesis.... 119
`[/1 A. Michael Rauth
`
`PART 2: CANCER BIOLOGY
`
`9. Properties ofMalignantCellsl39
`RonaldN. Buick and [an F. Tarmac/c
`
`10. CellProliferation.............................................................154
`
`Ian F. Tarmac/c
`11. Metasta51sl78
`Richard P. Hill
`12. TumorMarkers..................................._,................._.......196
`AaronMal/cirz
`
`13 Hormonesand Cancer. 207
`DonaldJ Sutherland and Betty G. Mobbs
`
`I
`
`(
`V
`
`1
`
`I
`
`\
`
`|
`
`|
`
`l
`.
`1
`
`I
`
`i
`
`

`

`
`
`vi
`
`CONTENTS
`
`14. ImmunologyandImmunotherapyofCancer....._._.........._......_._...._.._..
`Richard G. Miller and Ian F. Tarmock
`
`232
`
`PART 3: BIOLOGY UNDERLYING CANCER TREATMENT
`
`15. Cellular Basis of Radiotherapy .....
`Richard P. Hill
`
`. 259
`
`16. Experimental Radiotherapy............................_......_._.._..._...__._
`Richard P. Hill
`
`276
`
`17. Biological Properties of Anticancer Drugs. .
`Ian F. Tarmac/c
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`_
`
`.
`
`.
`
`_
`
`.
`
`.
`
`_
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`. ...
`
`. 302
`
`18. PharmacologyofAnticancerDrugs....._.__.............._...._._........._.....
`Charles Erlichmarz
`
`19. Experimental Chemotherapy......................_....._._.__..._._._.._.._._.
`Ian F. Tarmac/c
`
`317
`
`338
`
`20. Hyperthermia andPhotodynamic Therapy
`Richard P. Hill
`
`. 360
`
`21. Guide to Studies of Diagnostic Tests, Prognosis, and Treatment .....................
`Norman F. Boyd
`
`379
`
`Glossary.........._........_.........
`
`Index
`
`_ 395
`
`. 403
`
`

`

`
`
`19
`
`Experimental Chemotherapy
`
`Ian F. Tannock
`
`
`
`19.1 INTRODUCTION
`19.1.1 Current Status of Chemotherapy
`19.1.2 Therapeutic Index
`19.1.3 Relationship Between Tumor Remission and Cure
`
`19.2 DRUG RESISTANCE
`19.2.1 Mechanisms of Drug Resistance
`19.2.2 Genetic Basis of Drug Resistance
`19.2.3 Resistance to Methotrexate
`19.2.4 Multiple Drug Resistance
`19.2.5 Topoisomerases and Drug Resistance
`19.2.6 Glutathione and Drug Resistance
`
`19.3.3 Directed Drug Delivery
`19.3.4 Drugs That Cause Differentiation
`19.3.5 Anti-angiogenesis
`19.3.6 Drugs for Hypoxic and Nutrient-deprived Cells
`19.3.7 High-Dose Chemotherapy with Bone-Marrow
`Transplantation
`
`19.4 TREATMENT WITH MULTIPLE AGENTS
`19.4.1 Influence on Therapeutic Index
`19.4.2 Synergy and Additivity
`19.4.3 Growth Factors and Other Modifiers of Drug Activity
`19.4.4 Radiation and Drugs
`
`19.3 EXPERIMENTAL APPROACHES TO IMPROVEMENT
`OF CHEMOTHERAPY
`19.3.1 Adjuvant Chemotherapy
`REFERENCES
`19.3.2 Antimetastatic Drugs
`
`
`19.5 SUMMARY
`
`19.1 INTRODUCTION
`
`19.1.1 Current Status of Chemotherapy
`
`In current clinical practice chemotherapy is used in the
`following ways: (a) as the major curative modality for
`a few rare types of malignancies, such as Hodgkin’s dis-
`ease and other lymphomas, acute leukemia in children,
`and testicular cancer in men; (b) as palliative treatment
`for many types of advanced cancers; (c) as adjuvant
`treatment before or after local treatment (usually sur-
`gery) for primary disease, with the aim of eradicating
`occult micrometastases; and (d) in combination with
`other modalities (usually radiotherapy) in an attempt to
`improve their therapeutic effects.
`Improvements in clinical chemotherapy have de-
`pended largely on the use of drugs in combination.
`Some drugs have been combined because there is a the—
`oretical or experimental basis for expecting synergistic
`interaction through their known mechanisms of action
`either at the molecular level or because of their comple-
`mentary effects on cell-cycle kinetics. Synergy does not
`lead, however, to therapeutic benefit unless the interac-
`tion between drugs is tumor specific (section 19.4.1).
`
`The most important factors underlying the successful
`use of drugs in combination are (a) the ability to com-
`bine drugs at close to full tolerated doses with additive
`effects against tumors and less than additive toxicities to
`normal tissues, and (b) the expectation that drug com-
`binations will include at least one drug to which the tu-
`mor is sensitive.
`
`19.1.2 Therapeutic Index
`
`All anticancer drugs have toxicity as well as antiturnOr
`effects, and toxicity to normal tissues limits the dose 0f
`drugs that can be given to patients. The relationship be-
`tween probability of a biological effect of a drug and ad-
`ministered dose is usually described by a sigmoid cuI‘Ve
`(Fig. 19.1). If the drug is to be useful, the curve describ'
`ing probability of antitumor effect (e.g., complete clin-
`ical remission) must be displaced toward lower doses as
`compared to the curve describing probability of millor
`toxicity to normal tissues (e.g. , myelosuppreSSion lead'
`ing to infection). Therapeutic index (or therapeutic ra-
`tio) may be defined from such curves as the ratio of the
`doses required to produce a given probability of tOX‘
`
`338
`
`

`

`EXPERIMENTAL CHEMOTHERAPY
`
`339
`
`§
`3
`
`o g
`
`S g
`
`b) Normal tissue
`toxicity
`
`
`
`
`Therapeuticindex
`
`l
`ea) TD-05
`l
`ED-50
`
`I
`I
`I
`ED-50 TD-05 TD-5o
`Dose of Drug
`
`| I
`
`1.0
`
`g
`
`it? o 5
`E
`”L
`
`°
`
`Figure 191. Schematic relationships between dose of a drug
`and a the probability of a given measure of antitumor effect,
`and b, the probability of a given measure of normal—-tissue tox-
`icity. The therapeutic index might be defined as the ratio of
`doses to give 50% probabilities of normal-t-issue damage and
`antitumor effects. However, if the endpoint for toxicity is se-
`vere (eg., sepsis due to bone-marrow suppression), it would
`be more appropriate to define the therapeutic index at a
`lower probability of toxicity (e.g, TD-05/ED-5-0.)
`
`icity and antitumor “effect Therapeutic indexIn Fig-
`ure 19.1 might be represented by the ratio of the 5%
`level of probability of severe toxicity (sometimes referred
`to as Toxic Dose-05 or TD——05), and the 50% probabil-
`ity of antitumor effect (i. e. ,effective dose 50 or ED-~50).
`Any stated levels of probability might be used, and the
`appropriate endpoints of tumor response and toxicity
`will depend on the limiting toxicity of the drug, the1n-
`tent of treatment (i e., cure versus palliation), and on
`whether treatment is given to a patient or an experimen-
`tal animal However, dose—response curves similar to
`those of Figure 19.1 have been defined rarely for drug
`effectsin man.
`
`Improvement1n the therapeutic indexis the goal of
`experimental chemotherapy The concept emphasizes
`that any modification1n treatment that leads to in-
`creased killing of tumor cells1n tissue culture or animals
`must be assessed forIts effects on critical normal tissues'
`prior to therapeutic trials.
`
`a “surgically confirmed complete remission” may be
`compatible with the presence of a large number of tu-
`mor cells. Tumor cure requires eradication of all tumor
`cells that have the capacity for tumor regeneration. The
`proportion of such stem cells among those of the tumor
`population18 unknown (see chapter 9, section 9. 2), but
`clinical and even surgically confirmed complete remis-
`sions are compatible with the presence of a substantial
`residual population of tumor stem cells. Attainment of
`complete remission is but a small step toward tumor
`cure.
`
`For many drugs the relationship between cell survival
`and dose is close to exponential, so that a constantfrac-
`tion of the cells (rather than a constant number) is killed
`by a given dose of drug (chapter 17, section 17.2.4).
`Drugs are usually given in sequential courses, with dos-
`age and schedule limited by normal-tissue tolerance.
`Some repopulation of tumor cells may take place be-
`tween courses, so that the number of tumor cells in a
`
`drug-sensitive tumor may change with time during a
`course of chemotherapy, as illustrated in Figure 19.2.
`In this example, each course of drug kills 90% of the tu-
`mor cells, and starting from a large (100 g) tumor, com-
`plete clinical remission is achieved after three courses.
`Note that a further six to ten courses (depending on the
`prevalence of tumor stem cells) would be required to
`achieve cure. Realization of the need to continue aggres-
`sive treatment during complete remission, demonstrated
`originally by the experimental work of Skipper and his
`
`I2
`
`lc
`
`l l l I l I l
`
`'0
`
`I09
`
`
`
`2 3.
`l0 §1:.hi
`I0
`1?,
`we 5
`I. g
`'0 ,3
`
`"Complete remission"
`
`f
`
`l
`Response
`
`l
`i
`Drug resistance
`
`a
`g '0 Treatmenisl
`
`E
`3IO
`
`
`
`Time (months)
`
`"Tumor.
`CUIL
`
`19.1.3 Relationship Between Tumor
`Remission and Cure
`
`l
`
`For most solid tumors the limit of clinical and/or radio-
`logic detectionis about 1 g of tissue (~109 cells). If
`therapy can reduce the number of malignant cells below
`this limit of detection, the patient will be described as
`beingin complete clinical remission. Surgical biopsy of
`sites that were known to be previously involved with tu-
`mor may lower the limit of detection, but a pathologist
`is unlikely to detect sporadic tumor cells present at a fre-
`quency of less than 1in 1000 normal cells, so that even
`
`figure 19.2. Illustration of the relationship between tumor
`remission and cure. In this hypothetical example, treatment
`of a human tumor starts when it has 1011 cells (~100 g), and
`each treatment, given at monthly intervals, kills 90% of the
`cells present. This course of therapy leads to complete disap-
`pearance of clinical tumor. Drug resistance then develops, and
`the tumor grows despite continued treatment. Note that
`despite the attainment of a complete clinical response there
`are always at least 108 viable cells present, and that the
`reduction'In cell numbersIs small compared to that required
`for cure
`
`
`
`

`

`
`
`340
`
`THE BASIC SCIENCE OF ONCOLOGY
`
`led to success in the treatment of
`colleagues (1964),
`acute lymphoblastic leukemia in children, and subse-
`quently to cures in other tumors such as lymphomas.
`Unfortunately, for most solid tumors, a drug-resistant
`subpopulation emerges and leads to relapse, as shown
`in Figure 19.2.
`
`19.2 DRUG RESISTANCE
`
`19.2.1 Mechanisms of Drug Resistance
`
`Many types of cancer that occur commonly in man (e. g. ,
`colon cancer, lung cancer other than small-cell type)
`show only infrequent responses to treatment with anti-
`cancer drugs. This resistance to chemotherapy may be
`influenced by such factors as the proliferative state of
`the cells (chapter 17, section 17.2) and vascular access
`and penetration of drugs into tissue (section 19.3.6), but
`the most important factor is the intrinsic resistance of the
`tumor cells to available anticancer drugs. Other human
`tumors (e.g., breast cancer or small-cell cancer of the
`lung) often respond to initial treatment, but acquired re-
`sistance to further therapy usually prevents drug treat-
`ment from being curative. Thus intrinsic and acquired
`drug resistance are the major factors that limit the suc-
`cessful use of chemotherapy. A wide range of metabolic
`or structural properties of cells may lead to drug re-
`sistance, and some of the mechanisms underlying in-
`trinsic or acquired drug resistance are summarized in
`Table 19.1.
`
`Alkylating agents and cisplatin cause cellular dam—
`age by binding with DNA, leading to cross-linkages
`and breaks in DNA strands (chapter 18, section 18.3).
`Cells may be resistant to these drugs through a num-
`ber of mechanisms, including decreased cellular up-
`take, reduced drug activation, binding of alkylating
`species by sulfhydryl compounds such as glutathione
`(section 19.2.6), increased removal of drug adducts
`from DNA, and increased repair of DNA (see chap-
`ter 4, section 4.4 and chapter 17, section 17.2). Cross-
`resistance to several chemically unrelated drugs has
`been observed for naturally occurring and semisynthetic
`compounds such as doxorubicin and etoposide, due to
`common mechanisms of stimulated drug efflux from
`cells (section 19.2.4), or to decreased activity of the en-
`zyme topoisomerase II that allows conformational
`changes in DNA (section 19.2.5).
`‘A number of mechanisms may also lead to resistance
`to antimetabolite drugs. These mechanisms include im-
`paired drug transport into cells, overproduction or re-
`duced affinity of the molecular target, stimulation of
`alternative biochemical pathways, and impaired activa-
`tion or increased catabolism of the drug. The most
`widely studied of the antimetabolites is methotrexate,
`
`Table 19.1. Probable Mechanisms Associated with
`Resistance to Some Commonly Used
`Anticancer Drugs
`
`
`
`Mechanism Drugs
`
`Increase in proficiency Alkylating agents, cisplatin
`of repair of DNA
`
`Decrease in cellular
`uptake or increase in
`efflux of drugs
`
`Increase in levels of
`“target” enzyme
`
`Cisplatin, doxorubicin, etoposide
`melphalan, 6-mercaptopurine,
`methotrexate, nitrogen mustard,
`vinblastine, vincristine
`Methotrexate
`
`Alterations in “target”
`enzyme
`
`5-F1uorouracil, 6-mercaptopurine,
`methotrexate, 6—thioguanine
`
`Decrease in drug
`activation
`
`Increase in drug
`degradation
`
`Alternative bio-
`chemical pathways
`
`Inactivation of active
`metabolites by binding
`to sulfhydryl
`compounds
`
`Cytosine arabinoside, doxorubicin,
`5-fluorouracil, 6—rnercaptopurine,
`6-thioguanine
`
`Bleomycin, cytosine arabinoside,
`6-mercaptopurine
`
`Cytosine arabinoside
`
`Alkylating agents, cisplatin,
`doxorubicin
`
`Decrease activity of
`topoisomerase II
`
`Amsacrine, doxorubicin, etoposide
`
`
`and the varied mechanisms of resistance to this agent
`will be described as an example for other antimetabo-
`lites in section 19.2.3.
`
`19.2.2 Genetic Basis of Drug Resistance
`
`The following evidence suggests that many types of drug
`resistance are genetic in origin.
`
`1. Characteristics of drug-resistant cells (i.e. , their phe-
`notypes) are often stably inherited in the absence of
`the selecting drug.
`2. Drug-resistant cells are spontaneously generated
`with a rate that is consistent with known rates of ge'
`netic mutation.
`.
`
`3. Generation of drug-resistant cells is increased by ex‘
`posure to compounds (e.g. , ethyl methane sulfonate)
`that induce mutation. This property has been used
`to generate and select a large number of drug-resis‘
`tant mutants that have been used to study drug—resis—
`tant phenotypes (Fig. 19.3).
`4. Altered gene products have been identified in so!ne
`drug-resistant cells, and some of the genes have been
`cloned and sequenced.
`
`l'
`t
`
`1
`
`
`
`u.
`
`

`

`EXPERIMENTAL CHEMOTHERAPY
`EXPERIMENTAL CHEMOTHERAPY
`
`341
`
`MutagenQ Mutagen*9
`
`Mutagen
`
`Chinese Hamster
`Ovary Cells
`
`cultuye in low
`concentration of
`drug
`
`Culture surviving
`cells in intermediate
`concentration of
`drug
`
`Culture surviving
`cells'In high concentration
`of drug
`
`Select clones
`
`1
`
`Select clones
`
`l
`
`1
`
`
`
`Cells with increased levels of drug resistance
`
`figure 19.3. General method used for stepwise selection of drug-resistance mutant
`cells.
`
`
`
`
`
`5. Some drug-resistant phenotypes have been trans-
`ferred to drug-sensitive cells by transfer of genes,
`using techniques described in chapter 3, section 3.3.9.
`
`At least two types of genetic lesion may convey drug
`resistance: point mutation and gene amplification. Drug
`resistance due to point mutation may be expected to oc-
`cur in a single step that is not critically dependent on
`the concentration of drug used to select for resistance,
`and the degree of resistance should be quite stable. One
`of many well-characterized examples is mutation in the
`gene that codes for the enzyme HGPRT (hypoxanthine
`guanine phosphoribosyl transferase), which is neces-
`sary for activation of the drug 6-thioguanine. In con-
`trast to point mutation, gene amplification occurs in a
`stepwise manner, and drug-resistant cells are selected
`more rapidly by exposing cells to graded increases in the
`concentration of the selecting drug. The best-character-
`ized examples (Schimke, 1984; Bradley et al. , 1988) are
`amplification of the gene encoding dihydrofolate reduc-
`tase (DHFR), the target enzyme of methotrexate (sec-
`tion 19.2.3), and amplification of the Mir genes that
`encode P-glycoprotein (section 19.2.4).
`The frequency of occurrence of drug-resistant cells in
`a cell culture or tumor population may be estimated by
`plating the cells after exposure to the drug at a pharma-
`cologically achievable concentration and counting the
`resultant colonies. Typical spontaneous frequencies for
`the incidence of a variety of stable drug-resistant phe-
`notypes are in the range 10—7—10‘5. These frequencies
`are consistent with origin by spontaneous mutation, and
`definitive evidence for this origin has been obtained for
`a few drugs by performing a fluctuation test—a test de-
`vised by Luria and Delbruck (1943) to distinguish be-
`tween (a) mutation and selection and (b) induction by
`a drug, as the major cause of bacteriophage resistance
`
`in bacteria. The fluctuation test has been performed
`using only a few drugs (e.g., ouabain, L-asparaginase,
`and hydroxyurea), but has established mutation and se-
`lection as one major mechanism for generation of drug-
`resistant mutants.
`
`At least two other mechanisms might contribute to
`the generation of genetically based drug resistance.
`Many anticancer drugs (especially alkylating agents) are
`themselves mutagenic: treatment with such drugs may
`be expected to increase the rate of generation of mutant
`cells and to accelerate the development of resistance to
`themselves and to other drugs. Also, drugs that cause
`cell-cycle-progression delay during DNA synthesis may
`allow an increased rate of gene amplification. Thus,
`treatment with some drugs may stimulate the develop-
`ment of drug resistance.
`Second, there is evidence that different populations of
`cells may interact to influence the drug sensitivities of
`each other (e.g. , Heppner, 1984). This influence may
`involve sharing of drug metabolites or increased activa-
`tion of a drug by one of the cell populations. Cell fusion,
`the transfer of DNA between cells, or the uptake of
`DNA from dead cells are also potential mechanisms that
`could lead to horizontal transmission of drug resistance
`between mammalian cells.
`
`Generation of drug-resistant mutants among cells in
`human tumors has implications for planning optimal
`chemotherapy. Goldie and Goldman (1984) have dem-
`onstrated that the probability of there being at least one
`drug-resistant cell in a tumor population is dependent
`on tumor size (Fig. 19.4). This probability increases
`from near zero to near unity over a small range of tu-
`mor sizes (~6 doublings) with the critical size depend-
`ing on the rate of mutation to drug resistance. This
`effect, and others, imply a greater chance of cure if ther-
`apy is begun early, when only microscopic foci of tumor
`
`
`
`

`

`
`
`342
`
`THE BASIC SCIENCE OF ONCOLOGY
`
`0
`
`D
`/.’. —/'__.r0
`0/
`
`/.
`Mutation rate
`=l0‘“
`
`0
`
`Mutation rate
`= IO'6
`
`o
`
`.0ca
`
`9or
`
`.0a
`
`9N Probabilityofat/605/one
`
`drug-resisan/cell Ei-
`
`|
`
`no“
`:02
`Number of cells in tumor
`
`no“
`
`lo“
`
`Figure 19.4. Probability that there will be at least one drug-
`resistant cell in a tumor containing varying numbers of cells,
`based on rates of mutation of 10—6 (open symbols) and 10“4
`(closed symbols) per cell per generation. Note that this prob-
`ability increases from low to high values over a relatively short
`period in the life history of the tumor and that drug-resistant
`cells are likely to be established prior to clinical detection.
`(Adapted from Goldie and Coldman, 1984.)
`
`cells are present (see section 19.3.1). The Goldie—
`Coldman model also predicts a better therapeutic effect
`when two equally effective and non—cross-resistant
`drugs are alternated, rather than given sequentially,
`since this minimizes the emergence of cell populations
`that are resistant to both drugs.
`Although drug-resistant phenotypes in many types of
`cell have been shown to be due to mutation or amplifi-
`cation of genes, this could be due in part to methods
`used for selection of drug-resistant cells. Exposure of
`cells to mutagens, followed by selection in high concen-
`trations of drug (Fig. 19.3) may predispose to selection
`of cells with genetically based drug resistance. Exposure
`of cells to lower concentrations of drugs, without prior
`exposure to mutagens, often leads to cells that show
`transient resistance to drugs; indeed, transient resis-
`tance of some cells in the population may occur spon-
`taneously, without prior drug exposure (Cillo et al.,
`1989). Mechanisms underlying drug resistance that is
`unstable may include the transient amplification of genes
`or changes in patterns of DNA methylation or of other
`factors that influence gene expression (sometimes re-
`ferred to as “epigenetic”). Such changes in drug sensi-
`tivity might be expected to occur in vivo, since the cells
`are exposed to relatively low concentrations of drugs
`during cancer treatment. In one study, repeated treat-
`ment of tumor-bearing mice with alkylating agents led
`to high levels of resistance of the tumor in vivo, but no
`significant resistance was observed when cells derived
`from the tumors were exposed to the same drugs in cul-
`ture (Teicher et al., 1990). It was proposed that changes
`in the tumor cells led to changes in drug distribution in
`
`‘Qif‘l
`w" M
`
`vivo. Clinically important drug resistance is probably
`due to both genetic and nongenetic mechanisms.
`
`19.2.3 Resistance to Methotrexate
`
`Resistance to methotrexate may occur by several mech-
`anisms (Bertino et al., 1981; Fig. 19.5). Methotrexate
`is transported across cell membranes both by passive
`diffusion and by an energy-dependent active transport
`system. Drug-resistant cells may arise that have im-
`paired transport of methotrexate into the cell, probably
`due to point mutation (e.g. , Flintoff et a1. , 1976).
`Transport-deficient cells also show a decrease in poly-
`glutamation of intracellular methotrexate (Frei et al.,
`1984-); primary defects in polyglutamation or in intra-
`cellular binding could lead to decreased uptake of meth-
`otrexate in the drug-resistant mutants.
`A second type of mutation may lead to production of
`variant forms of DHFR, the target enzyme for metho-
`trexate. Variant enzymes have been found to retain ad-
`equate function for reduction of their normal substrate
`(dihydrofolate), but to have a greatly decreased affinity
`for methotrexate (e.g., Goldie et al., 1980).
`The most common mechanism leading to methotrex-
`ate resistance in cell lines and experimental tumors that
`are exposed to increasing concentrations of the drug is
`overproduction of DHFR from amplified genes. This
`process has been extensively characterized by Schimkc
`and his colleagues (e.g., Schimke, 1984-) and their ma-
`jor findings are:
`
`1. Selection for methotrexate resistance in mammalian
`
`cells by stepwise increase in drug concentration in
`
`Decreased formation
`of polyglutamaies
`
`Decreased transport
`'
`
`VarIant forms 0
`of MTX into cell
`
`\DH FR with low
`affinity for MTX
`
`
`|
`
`Methotrexate
`Polygluiamate
`Iormation
`(MTX)
`
`f
`
`Decreased binding
`of MTX to its turn:
`enzyme DHFR
`
`
`1/ Am/plified genes
`
`for DHFR on
`chromosomes or
`double minutes
`(leads to large
`increase in DHFR
`copies)
`
`Figure 19.5. Probable mechanisms underlying cellular resis-
`tance to methotrexate.
`
`

`

`EXPERIMENTAL CHEMOTHERAPY
`
`343
`
`Table 19.2. Drugs Associated with Cross-Resistance and
`Increased Expression of P-Glycoprotein
`
`Actinomycin D
`Colchicine
`Daunorubicin
`Doxorubicin
`
`Etoposide (VP-16)
`Mitoxantrone
`Puromycin
`Vinblastine
`
`Vincristine
`(Adriamycin)
`
`Epirubicin Vindesine
`
`that are selected for resistance to an individual drug of
`the group are cross-resistant to other members of the
`group but usually show the highest degree of resistance
`to the drug that was used for selection. Thus, this mech-
`anism of drug resistance shows some degree of specific-
`ity. Multidrug—resistant cells do not usually show major
`cross-resistance to antimetabolites or to alkylating
`agents, and increased sensitivity has been reported to a
`few drugs, including steroid hormones.
`When membrane proteins from multidrug—resistant
`cells are separated by electrophoresis, they are found
`to contain a glycoprotein whose molecular weight is
`about 170 kDa, which has been termed 'P-glycoprotein
`(Fig. 19.6).
`A direct correlation has been found between the con-
`
`tent of P-glycoprotein in the cell membrane and the de-
`gree of resistance to the selecting drug. Drug resistance
`can be transferred to drug-sensitive cells by transfection
`
`<8
`’0
`3' silage"
`v a? a? 5
`
`Origin...
`
`hi] P—glycoprotein
`'
`
`”Huh
`
`'50"
`IOOL
`
`50:
`
`l
`
`-
`
`
`
`%
`\.
`
`5‘
`
`k
`§
`
`the medium may lead to as many as 100—1000 cop-
`ies of the DHFR gene, and to high levels of drug
`resmtance.
`
`2. High levels of resistance to methotrexate cannot be
`obtained in a single-step selection process. Gene am-
`plification occurs in small steps.
`3. Resistance occurs from overproduction of the normal
`enzyme (although overproduction of variant forms of
`DHFR has also been observed).
`4-. Drug resistance and amplified genes may be either
`stable or unstable when cells are passaged in the ab-
`sence of the drug. Stable amplification is usually as-
`sociated with chromosomal location of the genes,
`seen as homogeneously staining regions in stained
`chromosome preparations (chapter 4-, section 4.3.1)
`Unstable amplification is usually associated with lo-
`cation of the genes in extrachromosomal chromatin
`structures known as double minutes. Both locations
`
`may be evident during selection for drug resistance.
`5. Gene amplification appears to take place by multiple
`replication ofthe DHFR gene (and flanking sequences)
`during the S phase of the cell cycle. Interruption
`of DNA synthesis in synchronized cells by drugs
`such as hydroxyurea, or by transient exposure to
`hypoxic conditions (Rice et al. , 1986), leads to an in-
`crease in the rate of gene amplification. Other anti-
`cancer drugs may increase resistance to methotrexate
`by gene amplification.
`6. Gene amplification and overproduction of DHFR
`have been observed in cells from human tumors
`treated with methotrexate.
`
`Although gene amplification has been studied most
`extensively in relation to methotrexate, there is increas-
`ing evidence for the importance of this mechanism in
`determining resistance to several other drugs, including
`5-fluorouracil and the multiple drug-resistance pheno-
`type described below.
`
`19.2.4 Multiple Drug Resistance
`
`Several investigators have observed that cells which are
`selected for resistance to one of a group of drugs (Ta-
`ble 19.2) may show cross-resistance to each of the other
`drugs in the group (for reviews, see Bradley et al. , 1988;
`Endicott and Ling, 1989). Thus, Chinese hamster ovary
`(CHO) cells selected for resistance to colchicine after
`prior treatment with a mutagen were found subse-
`quently to be cross—resistant to several anticancer drugs,
`including anthracyclines (e.g., doxorubicin) and vinca
`alkaloids (e.g. , Vinblastine), and to other drugs such as
`puromycin, an inhibitor of protein synthesis. Many of
`these drugs have quite different chemical structures, but
`most of them are derived from natural products. Cells
`
`IOIV
`
`Front.
`
`figure 19.6. Increased levels of P—glycoprotein in increasingly
`drug-resistant mutant Chinese hamster ovary cells derived
`from wild type (Aux B1 cells) by stepwise selection (as indi-
`cated in Fig. 19.3). Membrane components were separated by
`SDS gel electrophoresis, transferred to nitrocellulose paper,
`and stained using a radiolabeled hetero-antiserum. (Reprinted
`with permission from Ling, ”Genetic basis of drug resistance
`in mammalian cells.” In: Bruchovsky, Goldie (eds). Drugs and
`Hormone Resistance in Neoplasia. Copyright 1982, CRC Press,
`Boca Raton, FL.)
`
`
`
`

`

`
`
`344
`
`THE BASIC SCIENCE OF ONCOLOGY
`
`of DNA from drug-resistant cells or with cloned genes
`(known as mdr, or multidrug resistance genes) that en-
`code P-glycoprotein, demonstrating a cause-and-effect
`relationship between the presence of P-glycoprotein and
`drug resistance. P-glycoprotein is present at low concen-
`tration in the cell membrane of drug-sensitive cells, and
`drug resistance is most often due to amplification of mdr
`genes with a consequent increase in the production of
`P-glycoprotein. This effect is analogous to the amplifi-
`cation of the DHFR gene, which leads to resistance to
`methotrexate (section 19.2.3). In some multidrug resis-
`tant cells, there is increased transcription of Mr mRNA,
`without amplification of mdr genes.
`The mechanism underlying this type of multidrug re-
`sistance is reduced accumulation of drugs inside the cell.
`Studies using isotopically labeled drug (Fig. 19.7), or
`fluorescent compounds such as doxorubicin, have dem-
`onstrated a decrease in the net rate of uptake, and more
`rapid efflux, of drug from drug-resistant cells. There is
`evidence in some cells that P-glycoprotein may act as a
`permeability barrier to drug uptake, but its major func-
`tion appears to be as an energy-dependent efflux pump
`which extrudes a variety of unrelated compounds from
`cells (Bradley et al., 1988).
`Genetic analysis has demonstrated that P-glycopro-
`teins are produced by a small family of homologous ma’r
`genes, which have been cloned and sequenced from hu-
`man, hamster and murine cells (Gottesman and Pastan,
`1988; Raymond and Gros, 1989; Endicott and Ling,
`1989). Two homologous linked genes (ma’rl and W112
`have been located to chromosome 7 in human cells, but
`only the protein product of ma'rl is associated with drug
`
`resistance. Three ma’r genes have been identified in
`mouse and hamster cells, two of which appear to encode
`a functional P—glycoprotein. The sequences of the hu-
`man and rodent genes show about 80% identity, and
`each gene contains two homologous halves, suggesting
`that they were produced originally by duplication of a
`‘ smaller gene. Small variations in sequence lead to pref-
`erential resistance to different drugs (Safa et a1. , 1990).
`Sequencing of the genes has allowed determination of the
`amino acid composition of the corresponding proteins,
`and the location ofhydrophilic and lipophilic components
`has allowed modeling of the orientation of P-glycopro-
`tein in the cell membrane (Fig. 19.8). This model sug-
`gests that there are 12 transmembrane domains, with 2
`internal ATP-binding sites. P-glycoprotein has signifi-
`cant homology with a number of ATP—binding transport
`proteins found in bacteria, suggesting a general role for
`this type of protein in the energy-dependent efflux of a
`variety of agents from cells.
`Use of monoclonal antibodies against P-glycoprotein,
`or of specific genetic probes, that can bind to mdr
`mRNA in northern blots (chapter 3, section 3.3.4), has
`allowed quantitation of P—glycoprotein, or its mRNA,
`in cells from a variety of tumors and normal tissues.
`P-glycoprotein is present at high levels in the normal hu—
`man kidney and adrenal gland; at intermediate levels in
`lung, liver, colon, and rectum; and at low levels in most
`other ti