PERSPECTIVES
`
`Factors determining
`cellular mechanisms of
`resistance to antimitotic
`drugs
`
`Fernando Cabral
`
`Department of Integrative Biology and Pharmacology, University of Texas Medical
`School, Houston,TX, USA
`
`Abstract With the rapidly expanding use of paclitaxel and
`related taxanes to treat malignant diseases, comes the
`realization that development of resistance to this class of
`agents will become an increasingly significant clinical problem.
`Studies have indicated that acquisition of resistance to the
`cytotoxic action of these drugs can occur by limiting the drug’s
`ability to accumulate in cells, altering the stability of cellular
`microtubules, diminishing the drug’s ability to bind tubulin, or
`varying the expression of specific tubulin genes.This review will
`critically evaluate the selection methods used to generate drug
`resistant mutants in tissue culture and focus on the various
`factors that determine which resistance mechanisms are most
`likely to be encountered. It is anticipated that clinical drug
`resistance will be complicated by pharmacokinetic
`considerations and variability among individuals, but that
`underlying genetic mechanisms will be similar to those found in
`culture. © 2001 Harcourt Publishers Ltd
`
`It is generally accepted that tumor cell resistance to
`chemotherapeutic drugs represents the single most signific-
`ant reason for the failure of drug therapy to cure cancer.1
`Paclitaxel should prove no different in this regard. In this ar-
`ticle I will discuss approaches currently being used to study
`drug resistance while emphasizing that emerging mechan-
`isms are highly influenced by the selections used to obtain
`resistant cell lines.Throughout the text I will use paclitaxel
`as the prototype for the increasingly broad class of drugs
`known as taxanes, but will include other antimitotic drugs as
`needed to illustrate specific points. This review is not
`intended to be comprehensive; instead I will cite a few
`experiments to illustrate various principles or mechanisms.
`Although the discussion will focus on mechanisms of res-
`istance to antimitotic drugs, some of the general principles
`may have relevance to other drug classes as well. I apologize
`in advance to those whose work is not included here. It is
`not meant to diminish the importance of their work, but
`rather to limit the scope of the review. For a more compre-
`hensive summary of the literature, the reader is referred to
`another recent review.2
`Tumor cells from patients are frequently very hetero-
`geneous, slow growing, and difficult to culture. For these
`reasons most information about drug resistance mechanisms
`has come from studying established cell lines in culture.
`Although these are far removed from a true in vivo situation,
`the ease with which resistant cells can be generated
`and studied, the ability to maintain tight controls, and the
`
`flexibility with which treatment protocols can be varied,
`combine to make cell culture the system of choice for the
`study of drug resistance mechanisms. It should be remem-
`bered, however, that mechanisms discovered in cell culture
`may not always represent the most commonly encountered
`mechanisms in patients undergoing treatment. Nonetheless,
`cell culture systems are a good place to begin examining the
`genetic basis and relative frequencies of potential cellular-
`based mechanisms of resistance.
`
`INFLUENCE OF THE SELECTION METHOD
`
`One of the biggest advantages of the cell culture model for
`drug resistance, namely the flexibility to manipulate the drug
`treatment protocol, can also lead to one of its biggest pitfalls.
`A variety of selection protocols for isolating drug resistant
`mutants has been employed, but seldom have the implica-
`tions of choosing a particular method of selection been dis-
`cussed. Most studies have used multiple step procedures in
`which cells are initially selected under low, minimally toxic
`drug concentrations, but are then exposed to many further
`stepwise increases until cells with very high levels of resist-
`ance are obtained.While this method offers the advantage of
`producing biochemical changes that are relatively easy to
`detect and study, one must recognize that the method is
`biased in favor of resistance mechanisms that are capable of
`producing high levels of resistance. Furthermore, it is likely
`that multiple genetic changes have contributed to the
`drug resistance, but the individual contributions are not
`always easy to sort out. In many cases, they are not even
`acknowledged.
`The problem of bias is significant because chemother-
`apeutic drugs are typically administered to patients in an
`amount that is close to the maximum tolerated dose, and so
`the clinician rarely has the option of increasing the drug con-
`centration when a patient relapses. For this reason, single-
`step selections with a concentration of drug only a few-fold
`higher than the minimal toxic dose should reveal resistance
`mechanisms that are most clinically relevant. At the same
`time, single-step selections should be less biased, i.e. mech-
`anisms that produce high and low levels of resistance should
`be retained, and the resulting phenotypes should be more
`easily ascribed to specific biochemical and genetic changes.
`An example of how selection methods can bias the kinds
`of mutants that are isolated is provided by resistance to
`antimitotic drugs. Most cell lines resistant to paclitaxel, a
`drug that stabilizes microtubules and blocks cells in mitosis,
`have been obtained using a multiple step procedure.2
`Typically, these cells are hundreds or even a thousand-fold
`resistant to paclitaxel and exhibit cross-resistance to a vari-
`ety of hydrophobic drugs with diverse mechanisms of
`action. Analysis of the molecular defect in these cells has
`revealed increased production of P-glycoprotein, an ATP-
`driven plasma membrane pump that actively extrudes
`hydrophobic drugs from the cell and is responsible for a
`major form of multidrug resistance.3 On the other hand,
`laboratories using selections that give lower levels of resis-
`tance have reported resistance mechanisms based on tubulin
`mutations that affect microtubule assembly and stability4–8 or
`binding of the drug to tubulin.9 Indeed, in our own studies
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`2001 Harcourt Publishers Ltd Drug Resistance Updates (2001) 4, 3–8
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`doi: 10.1054/drup.2000.0172, available online at http://www.idealibrary.com on
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`002009
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`AVENTIS EXHIBIT 2009
`Mylan v. Aventis, IPR2016-00712
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`
`Cabral
`
`using single-step selections for paclitaxel resistance, most
`resistant cell lines had altered microtubule stability and less
`than 10% were multidrug resistant.8
`In retrospect, the predominance of the multidrug resist-
`ance mechanism in multiple step, but not single step, selec-
`tions was predictable. Tubulin is a highly conserved and
`tightly regulated protein that is essential for cell survival;
`therefore, only subtle mutations that minimally disturb its
`ability to assemble into microtubules are retained. Such
`mutations produce modest effects on drug resistance, and
`so, cells with tubulin alterations are typically only 2–3-fold
`resistant to the selecting agent. P-glycoprotein is under no
`such constraints. It is a non-essential protein (to a tumor cell)
`and its level in the plasma membrane is not highly regulated.
`Multiple step selections producing high levels of resistance
`therefore retain cells with highly elevated production of nor-
`mal or mutant P-glycoprotein because the cells suffer no
`adverse effects. Clearly then, multiple step selections favor
`multidrug resistant cells over those with tubulin alterations,
`whereas single step procedures exhibit no such bias.
`A second problem with multiple step selections is the dif-
`ficulty in ascribing a specific genetic or biochemical change
`to the drug resistance phenotype. The growth of cells
`between rounds of selection allows the introduction of mul-
`tiple mutations that may be contributing to drug resistance.
`Thus, identification of a single biochemical change may not
`be sufficient to define what could be a complex mechanism
`of resistance or overlapping mechanisms of resistance.
`Studies in mouse macrophage J774.2 cells selected to grow
`in high concentrations of paclitaxel, for example, identified a
`clear increase in P-glycoprotein as the mechanism of resist-
`ance.10 Later studies, however, reported that these cells grew
`better in the presence of paclitaxel than in its absence,11 a
`phenotype previously described among Chinese hamster
`ovary (CHO) cells with altered tubulin that had been
`selected for resistance in a single step.4,5,8 It thus appears that
`during selection, the J774.2 cells acquired tubulin alterations
`that affected assembly and subsequently acquired further
`mutations leading to increased production of P-glycoprotein
`to produce the complex phenotype.
`A more recent example of problems in interpretation that
`can be encountered in multiple step selections is provided
`by reports of increased b
`-tubulin production as a mechanism
`of paclitaxel resistance. Mammals express at least seven dis-
`tinct genes producing highly homologous b
`-tubulin isoforms
`that differ primarily in their carboxyl terminal sequences.12
`Biochemical studies have suggested that the various b
`-
`tubulin isoforms have differing assembly and drug binding
`properties.13 It would therefore be reasonable to expect that
`altered production of specific b
`-tubulin isotypes might allow
`a cell to become resistant to specific antimitotic drugs. A
`number of studies support this idea. For example, various
`cell lines have been exposed to increasing concentrations of
`paclitaxel and then analyzed for b
`-tubulin expression using
`quantitative PCR. This approach has led to publications
`reporting an increase in b 2-tubulin in J774.2 cells,14 an
`increase in b 1, b 2, b 3, and b 4a-tubulin in human lung cancer
`cells,15 an increase in b 1, b 3, and b 4a in human ovarian
`tumors,15 and an increase in b 4a in human leukemia cells.16
`Additionally, an increase in b 3 and b 4a was found in human
`
`prostate cancer cells selected for resistance to estramustine,
`a drug that inhibits microtubule assembly.17
`Although several independent laboratories have reported
`these changes, there is as yet no compelling evidence that
`altered tubulin expression is sufficient to produce resistance
`to antimitotic drugs. First, of the seven isotypes of b
`-tubulin
`produced in vertebrates, four have been implicated in res-
`istance to paclitaxel, a seemingly high number. Second,
`increased expression of b 3 and b 4a tubulin has been implic-
`ated in resistance to paclitaxel and to estramustine even
`though the two drugs have opposing actions on micro-
`tubules and bind to different sites on tubulin. Third, one
`study reported changes in tubulin expression in human sar-
`coma cells, but these did not correlate with paclitaxel resist-
`ance.18 Finally, transfection and overexpression of b 1, b 2 or
`b 4b-tubulin was found to have no effect on paclitaxel resist-
`ance in CHO cells.19 How is one to explain these conflicting
`results? One possibility is that tubulin expression varies from
`cell to cell in heterogeneous populations and that isolation
`of a subclone with altered expression is unrelated to drug
`resistance. A second and perhaps more likely possibility is
`that the increased expression of tubulin is linked to some
`other change such as a mutation in the highly expressed
`tubulin gene, and the mutation is actually responsible for the
`resistance.Whatever the eventual explanation, these ambigu-
`ities in interpretation point out the need to firmly establish
`a cause and effect relationship between any biochemical
`change that is identified and the drug resistance phenotype
`of the mutant cells.
`
`CHOICE OF DRUG
`
`The bias introduced in multiple step selections suggests that
`the prominence of multidrug resistance may be overstated
`in the literature: a conclusion supported by the observation
`that paclitaxel has been highly successful in patients previ-
`ously treated with anthracycline antibiotics, which are well
`known substrates for the P-glycoprotein pump.20–22This argu-
`ment, however, is not meant to diminish the potential impor-
`tance of multidrug resistance mechanisms even in single
`step selections. Our laboratory has examined the prevalence
`of tubulin alterations versus multidrug resistance mecha-
`nisms in single step selections using various antimitotic
`drugs.8,23 The studies (Table 1) demonstrated that the preval-
`ence of a particular drug resistance mechanism is highly
`dependent on the selecting drug. Despite the fact that all the
`drugs tested are affected by the multidrug resistance mech-
`anism, the frequency with which this mechanism was seen
`
`Table 1 Frequency of MDR in single step selections using the
`indicated drug
`
`Drug
`
`MDRa
`
`Tubulinb
`
`%MDR
`
`Colchicine
`Vinblastine
`Paclitaxel
`
`17
`17
`10
`
`3
`5
`129
`
`85
`77
`8
`
`aNumber of cell lines with the multidrug resistance phenotype. bNumber
`of cell lines with properties indicating a tubulin alteration.
`
`4
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`
`Resistance to antimitotic drugs
`
`in single step selections varied from a high of 75–85% for vin-
`blastine and colchicine, to a low of 8% for paclitaxel.
`Although the reasons for this variability are uncertain, one
`plausible explanation is that tubulin mutations conferring
`paclitaxel resistance destabilize microtubule structure and
`are relatively common. On the other hand, tubulin mutations
`that confer resistance to colchicine and vinblastine would
`need to stabilize microtubule structure and these are rel-
`atively rare.The lesson from these studies is that one cannot
`a priori assume that a resistance mechanism identified for a
`particular drug will apply equally to all drugs within the
`same class.
`
`CHOICE OF CELLS
`
`The cell line used in studies of drug resistance may also influ-
`ence the kinds of mutants that are ultimately obtained.
`Differences in membrane properties, tubulin composition,
`and other less obvious factors may all combine to determine
`the relative frequencies with which various mechanisms are
`seen.As already mentioned, most human tumor cell lines are
`relatively heterogeneous and it is likely that significant vari-
`ability in biochemical and genetic properties exists from one
`cell to the next.This can even be a problem with well estab-
`lished cell lines and can complicate interpretation of mutant
`phenotypes. For this reason, it is wise whenever possible to
`clone cells before beginning a mutant selection as this will
`minimize the influence of genetic drift and make the assign-
`ment of a biochemical change to the phenotype of the
`mutant less ambiguous.
`Our studies have used Chinese hamster ovary (CHO) cells
`for mutant isolation.This is a well established, stable cell line
`that is easily cloned and has been well studied. CHO cells
`express 3 b
`-tubulin isoforms (b 1, b 4b, and b 5) with relative
`abundances of 70%, 25%, and 5% respectively.24,25 To date,
`every b
`-tubulin mutation we have identified in cells selected
`for resistance to antimitotic drugs was found in the b 1 iso-
`form. This may not be surprising because b 1 is the most
`abundant isoform and is therefore positioned to have the
`greatest effect on microtubule assembly. More recent studies
`using site directed mutagenesis and transfection of b
`-tubulin
`cDNA to explore the kinds of changes that allow acquisition
`of paclitaxel resistance, however, indicate that some highly
`toxic mutations can confer resistance but only when
`expressed at low levels (F. Cabral, unpublished studies). It is
`therefore possible that further sequencing of mutant cell
`lines will uncover some of these more toxic mutations in less
`highly expressed tubulin genes.A tentative conclusion from
`this work is that less disruptive mutations will be found in
`highly expressed genes and the more disruptive mutations
`will be found in less highly expressed genes.Thus, the kinds
`of mutations that are found are likely to be influenced by the
`tubulin composition of the cell line used for mutant selec-
`tion.
`It should be noted that our CHO cells with mutant tubu-
`lin are resistant to antimitotic drugs because of altered stabil-
`ity of their microtubules.26,27 Moreover, recent selections
`using human KB3 cells produced mutant cell lines with
`properties that mimic those we’ve seen in CHO cells
`(F. Cabral, unpublished studies). Despite the isolation of
`
`hundreds of drug resistant cells, we have to date not identi-
`fied any mutants that have altered drug binding even though
`such changes are common in lower eukaryotes.28,29 This dis-
`crepancy can be explained by the observation that antimitotic
`drugs poison microtubule assembly at substoichiometric con-
`centrations.30 Thus, a decrease in drug binding affinity can
`confer resistance in yeast because they grow as haploid cells
`that express a single b
`-tubulin gene. Mammalian cells, on the
`other hand, are diploid and express multiple tubulin genes.
`In this case, a mutation that decreases the drug binding affin-
`ity will affect only a small portion of the total tubulin, leaving
`sufficient wild-type tubulin to bind the drug with normal
`affinity and poison microtubule assembly. In short, decreased
`drug binding affinity is a recessive phenotype that should
`not be observed in mammalian cells.
`Contrary to this expectation, mutant 1A9 cells have been
`reported to contain altered b 1-tubulin with decreased bind-
`ing of paclitaxel.9 This human ovarian carcinoma cell line
`was subjected to several rounds of exposure to increasing
`concentrations of paclitaxel leading to the isolation of two
`mutant cell lines with approximately 20-fold resistance to
`the drug.Although isolation of these mutants would appear
`to contradict the idea that decreased drug binding is a reces-
`sive phenotype, further analysis of the cells revealed that the
`wild-type allele of the b 1-tubulin gene was not expressed.9
`Because the authors estimate that the b 1 isoform accounts
`for about 85% of the total, most of the b
`-tubulin in the cells is
`altered; and this may explain why drug binding alterations
`were recovered in their selections. Subsequent selection of
`cells resistant to epothilone A or B by the same authors again
`led to the isolation of drug binding mutations.31 These stud-
`ies present a dramatic example of how the choice of a par-
`ticular cell line can influence the mechanism of resistance
`that is seen.
`
`CLINICAL PERSPECTIVES
`
`Taking into account the previous discussion, one might ask
`what kind of mutant tumor cells are likely to be found in
`patients undergoing therapy with one of the antimitotic
`drugs. Given that a patient is likely to receive a single con-
`centration of drug, the single-step selection model should be
`the best predictor for the relative frequencies with which
`the various drug resistance mechanisms are encountered.As
`already pointed out, the most frequently encountered mech-
`anism will depend on which drug is being administered. For
`vinca alkaloids and many other drugs that inhibit micro-
`tubule assembly, it is expected that multidrug resistance will
`predominate. For paclitaxel, epothilone, and other drugs that
`promote microtubule assembly, on the other hand, tubulin
`mutations should be seen most frequently.The development
`of methods to overcome multidrug resistance and the identi-
`fication of new antimitotic drugs that are not affected by this
`phenomenon have all been extensively discussed in the liter-
`ature and will not be reviewed here. It should be noted,
`however, that as methods to circumvent multidrug resistance
`become adopted, tubulin alterations as a mechanism of res-
`istance will become increasingly prevalent.
`Tubulin mutations can confer resistance to antimitotic
`drugs by at least two mechanisms. One of these, decreased
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`Cabral
`
`drug binding, is rarely if ever seen in a well behaved cell line.
`Tumors, however, are anything but well behaved and fre-
`quently exhibit genomic instability.32 It is therefore possible
`that some fraction of large tumors will have become func-
`tionally haploid at the b
`-tubulin locus and thereby be able to
`survive cancer therapy by acquiring mutations that decrease
`drug binding.Two considerations suggest that such mutants
`will be infrequently encountered. First, this phenotype
`requires at least two independent changes (haploidization of
`the b
`-tubulin locus and mutation of the expressed b
`-tubulin).
`Second, these mutants have only been reported in multiple
`step selections to approximately 20-fold resistance. Neverthe-
`less, if this mechanism is encountered in a patient, the resis-
`tance should exhibit some specificity for the drug used in the
`selection. In cell lines with decreased binding of paclitaxel,
`for example, only low cross-resistance to epothilones was
`reported even though both drugs act by stabilizing micro-
`tubules and bind to the same region of tubulin.9 Similarly, cell
`lines with altered binding of epothilones exhibited much
`lower cross-resistance to paclitaxel.31
`A second mechanism by which tubulin alterations can
`confer resistance is through changes in microtubule stability.
`This phenotype can arise from a single mutation in either a
`-
`or b
`-tubulin6,26 and should therefore be encountered more
`frequently than drug binding mutations. As already men-
`tioned, this is the mechanism most commonly found in sin-
`gle step selections for paclitaxel resistance.8 The mechanism
`is most easily understood by considering that microtubules
`are metastable, highly dynamic structures that can only func-
`tion within a limited range of stability (Fig. 1). This limited
`stability is reflected in the fact that the microtubule polymer
`exists in a steady state with free heterodimers. In CHO cells
`only 38% of the cellular tubulin is in the polymer pool.33
`Drugs like vinblastine that destabilize microtubules decrease
`the amount of polymer. These drugs become toxic when
`their concentration reduces the amount of microtubules to
`the point they become nonfunctional (point L, Fig. 1). Drugs
`like paclitaxel, on the other hand, increase the amount of
`polymer and become toxic at concentrations that abrogate
`the ability of the cell to control tubulin polymerization
`(point H, Fig. 1). Cells may become resistant when tubulin is
`altered in a way that counteracts drug action.Thus, mutations
`that stabilize microtubules confer resistance to vinblastine
`
`and other inhibitors of polymerization because more drug is
`needed to reduce microtubule stability below point L.
`Mutations that destabilize microtubules confer resistance to
`paclitaxel and other agents that promote assembly because
`more drug is needed to raise microtubule stability beyond
`point H.
`The same mutations that confer resistance to one drug
`may make the cell more sensitive to another. For example,
`we frequently find that cells selected for resistance to a drug
`that enhances microtubule stability (e.g. paclitaxel) are
`cross-resistant to other drugs that enhance stability (e.g.,
`epothilones), but are more sensitive to drugs that destabilize
`the polymer (e.g. vinblastine). Conversely, cells that are resist-
`ant to drugs that destabilize microtubules are cross-resistant
`to all drugs that destabilize microtubules, but are more sensi-
`tive to paclitaxel, epothilone, and other microtubule stabiliz-
`ing drugs. Thus, it might be anticipated that patients who
`relapse following treatment with paclitaxel might be good
`candidates for follow-up therapy with vinblastine, but not
`epothilones.This prediction has not yet been well tested.
`One problem in applying these principles to patient man-
`agement is the difficulty in knowing the mechanism by
`which a patient has relapsed.Although good molecular tools
`are available to determine whether P-glycoprotein is ele-
`vated in resistant tumors, determining whether mutations
`exist in tubulin is far more challenging. Recent work, how-
`ever, suggests that development of methods to detect tubulin
`mutations in a cost effective way might be feasible.Although
`it is reasonable to think that a mutation destabilizing micro-
`tubule structure and conferring paclitaxel resistance can
`occur anywhere on b
`-tubulin, it has been reported that such
`mutations affect only three amino acids, all leucine, at posi-
`tions 215, 217, and 228 of the protien.34 This observation
`suggests that it should be possible to devise a diagnostic
`assay for these mutations and thereby provide the clinician
`with important information about the mechanism of resis-
`tance in a tumor and potential follow-up therapies that could
`be used to attack the malignancy.
`
`CAVEATS
`
`This review has focused on mechanisms of resistance likely to
`be encountered in a well behaved cell line in culture; but
`
`Fig. 1 Mechanism by which changes in microtubule stability confer resistance to antimitotic drugs. Function is maintained when
`microtubule stability falls between points L and H.Wild-type (WT) CHO cells have microtubule stability that leads to 38%
`assembly of the total tubulin. PTXR, extent of assembly in paclitaxel resistant cells;VLBR, extent of assembly in vinblastine
`resistant cells.
`
`6
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`Table 2 Potential mechanisms of resistance to antimitotic drugs
`
`Resistance mechanism
`
`Comments
`
`Cellular
`Increased drug efflux
`
`Altered tubulin assembly
`
`Altered tubulin synthesis
`
`Altered drug binding
`
`Pharmacological
`Increased drug metabolism
`Increased drug excretion
`Decreased delivery to tumor
`
`Cytokinetic
`
`Common for microtubule inhibitory
`drugs, less common for stabilizing
`drugs
`Common for microtubule stabilizing
`drugs, less common for inhibitory
`drugs
`Not proven as mechanism of
`resistance
`Common in haploid cells, rare in
`diploid
`
`e.g. poor vascularization of tumor;
`cells in pharmacological sanctuaries
`(CNS, testes, ovaries)
`
`e.g. drug not present at appropriate
`stage of cell cycle
`
`tumor cells are more heterogeneous and exist in much more
`complex environment. Thus, tumor cells in patients could
`escape therapy because of pharmacological considerations I
`have not addressed (Table 2). Problems in drug delivery to the
`tumor or metabolism of the drug are likely to vary from
`patient to patient and may lead to treatment failure. It should
`be easier, however, to circumvent these problems when they
`occur.The genetic changes leading to drug resistance are likely
`to affect all patients and may be more difficult to attack.
`I have also not addressed a growing body of literature
`indicating that changes in cell cycle parameters or apoptosis
`might be involved in drug resistance. While these kinds of
`changes may yet prove to be important stumbling blocks to
`successful chemotherapy, it is too soon to assess their signifi-
`cance in relation to the more specific mechanisms I have dis-
`cussed. It should be pointed out, however, that in our
`selections for resistance to antimitotic drugs, we have found
`no evidence for the existence of these alternative mecha-
`nisms at any appreciable frequency.
`Finally, I should point out that the changes I have dis-
`cussed assume continuous exposure to a single cytotoxic
`drug; but patients undergoing chemotherapy receive inter-
`mittent therapy with several drugs in combination. This
`might suggest that mechanisms capable of conferring resis-
`tance to different classes of drugs simultaneously should be
`most common in patients. As already mentioned, however,
`the observation that patients refractory to anthracycline
`treatment respond well to paclitaxel provides evidence that
`drug specific mechanisms of resistance may be more com-
`mon than is presently appreciated.
`
`CONCLUSION
`
`A large body of work has established the most common
`mechanisms of cellular resistance to antimitotic drugs. The
`
`Resistance to antimitotic drugs
`
`challenge in the future will lie in devising methods to detect
`these mechanisms in drug resistant human tumors, establish-
`ing their prevalence, and working out alternative therapies
`to circumvent their emergence. As already mentioned, the
`identification of tubulin mutations that confer resistance to
`paclitaxel and related drugs suggests that tools will become
`available in the next few years to begin screening patients
`for the presence of these genetic alterations. At the same
`time, it will become increasingly important to assess the role
`of pharmacological changes in mediating resistance. The
`inability to experimentally manipulate a patient population
`poses a serious obstacle to carrying out these studies; but the
`use of heterotransplanted tumors in rodents may provide a
`convenient model in which to determine the importance of
`pharmacokinetic alterations in drug resistance, and to test
`strategies for overcoming these kinds of changes. Given the
`rate at which new information is becoming available, it is
`possible to foresee a day in the near future when a patient
`will be evaluated for preexisting mechanisms of resistance,
`receive customized therapy based on that evaluation, be re-
`tested at regular intervals for the possible emergence of new
`mechanisms of resistance, receive an altered course of ther-
`apy to combat emerging resistant cells, and enjoy a more
`favorable outcome than is currently attainable.
`
`Acknowledgements
`I want to thank the people who have worked in my labor-
`atory for generating much of the work that was described
`and for stimulating discussions that led to many of the ideas
`that were expressed. Studies in my laboratory have been
`funded by generous support from the National Cancer
`Institute of the Public Health Service.
`
`Received 11 October, 2000; Revised 10 November, 2000;
`Accepted 10 November, 2000
`
`Correspondence to: Fernando Cabral PhD, Department of Integrative Biology
`and Pharmacology, University of Texas-Houston Medical School, P.O.Box
`20708, Houston,TX 77225, USA.Tel:+1 713 500-7485; Fax:+1 713
`500-7455; E-mail: fcabral@uth.tmc.edu
`
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`Cabral
`
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