Similarities and differences between the multidrug resistance phenotype of mammalian tumor cells and chloroquine resistance in Plasmodium falciparum

EXPERIMENTAL

PARASITOLOGY

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MINIREVIEW Similarities and Differences between the Multidrug Resistance Phenotype of Mammalian Tumor Cells and Chloroquine Resistance in Plasmodium falciparum STEVEN KARCZ AND ALAN F. COWMAN The Walter and Eliza Hall Institute of Medical Research, Melbourne 3050, Australia KARCZ, S., AND COWMAN, A. F. 1991. Similarities and differences between the multidrug resistance phenotype of mammalian tumor cells and chloroquine resistance in Plasmodium falciparum. Experimental Parasitology 73, 233-240. o 19% Academic press, IK.

INTRODUCTION

The chloroquine resistance (CQR) phenotype in Plasmodium falciparum has been likened to a form of multidrug resistance (MDR) found in some mammalian tumor cell lines. This similarity stems from two key features of both phenotypes which distinguish them from other forms of drug resistance. First, resistance to the cytotoxic compound in question is characterized by enhanced efthrx of cytotoxic drug in drugresistant cells when compared to their drugsensitive counterparts (Skovsgaard 1978; Fojo et al. 1985; Krogstad et al. 1987; Wellems et al. 1990). Second, sensitivity to a cytotoxic drug-resistant cells can be reinstalled by coadministration of one of a family of chemically distinct agents which are termed reversal or chemosensitizing drugs (Fojo et al. 1985; Rogan et al. 1984; Martin et al. 1987; Bitonti et al. 1988; Kyle et al. 1990). Thus, while the pharmacokinetic parameters governing drug/target cell interaction are clearly distinct in both CQR and MDR (and indeed among different antineoplastic drugs involved in MDR), it is possible that the underlying mechanisms giving rise to the two common phenotypic features of CQR and MDR listed above may be similar. In this review, we would like to focus on the phenotypic similarities between CQR in

P. falciparum and MDR in mammalian tumor cells and to discuss the relationship of mdr gene products (P-glycoproteins) to these phenotypes. Readers are also referred to a number of recent comprehensive reviews on aspects of CQR and MDR which are beyond the scope of this discussion (Gottesman and Pastan 1988; Endicott and Ling 1989; Dhir et al. 1990; Cowman and Foote 1990). MODULATION OF DRUG RESISTANCE

The similarity between CQR and MDR is particularly striking in the area of modulation of drug resistance. Accordingly, some antimalarial compounds such as chloroquine, quinine, and quinidine are capable of sensitizing MDR tumor cells (Rogan et al. 1984; Fojo et al. 1985; Mickisch et al. 1991), while some classical anticancer drugs such as daunomycin and vinblastine can render CQR P. falciparum susceptible to chloroquine (Krogstad et al. 1987). In addition, both CQR in P. falciparum and MDR in mammalian tumor cells can be modulated by a number of the same compounds. However, not all drugs capable of chemosensitizing MDR tumor cells are similarly efficacious for CQR P. falciparum. Nonetheless, the restoration of drug sensitivity in both CQR and MDR in the presence of chemomodulating agents is a result of enhanced retention of the cytotoxic com-

233 0014-4894/91$3.ocl Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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pound. It is clear that complete reversal of MDR in tumor cells can not always be achieved and many examples of incomplete reversal have been reported (Rogan et al. 1984; Klohs et al. 1986; Tsuruo et al. 1983; Beck et al. 1986). Also, measurements of drug accumulation in these experiments show that complete reversal of MDR in tumor cells is not always accompanied with an increased drug accumulation level comparable to that observed in drug-sensitive parental cells (Willingham et al. 1986). Similar observations have been made in the case of CQR in P. falciparum (Krogstad et al. 1987). A feature shared by both the MDR and CQR phenotypes is the rapid etllux of cytotoxic drugs so that the intracellular concentration does not reach cytotoxic levels. In the case of MDR in mammalian tumor cells, the P-glycoprotein has been clearly implicated (see Endicott and Ling 1989) but the effector molecule(s) involved in expulsion of chloroquine from resistant P. falciparum has not been identified. CQR P. falciparum isolates are able to expel 40 to 50fold more chloroquine than CQS cells and this process has a half-life of 1 to 2 min (Krogstad et al. 1987). The half-life of drug eMux from MDR tumor cells is difficult to measure as it is an extremely rapid process. Using a pulsed quench-flow apparatus to study transport characteristics of daunomytin in MDR tumor cells it was possible to show that greater than 50% of daunomycin efflux occurred within 0.1 set, while in sensitive cells there was no substantial efflux (Cano-Gauci et al. 1990). The transport process of drugs in MDR tumor cells is a much more rapid process than occurs in CQR P. falciparum and this is perhaps not unexpected as the tumor cell lines are usually much more resistant to the cytotoxic drugs than is the case in CQR. There are also a variety of other factors which may influence the accumulation and expulsion of cytotoxic drugs in MDR tumor cells (Demant et al. 1990).

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The molecular basis of the reversal of MDR in mammalian tumor cell lines is only beginning to be appreciated and is thought to derive from direct competition between the chemosensitizing agent and the antitumor drug for a limited number of binding/ translocation sites residing within the tertiary structure of an active efflux agency (P-glycoprotein or P-gp) (Safa et al. 1990; Greenberger et al. 1990; Bruggemann et al. 1989). The P-glycoprotein homologues (Pghs) of P. falciparum, on the other hand, remain functionally uncharacterized and, as such, a direct link between any aspect of the CQR phenotype and the function of pfmdr gene products has not been established. Nonetheless, a direct approach in the functional analysis of P. falciparum Pghs will not only allow us to understand their normal physiological role but also to directly address the issue of whether the similarities between the MDR phenotype in mammalian tumor cells and chloroquine resistance in P. falciparum extends to a role for these transport molecules. CROSS-RESISTANCE

MDR in mammalian tumor cells is a unique phenomenon where cell lines displaying this phenotype have been selected for resistance to a single cytotoxic drug and similtaneously display cross-resistance to a wide array of structurally unrelated cytotoxic drugs (see Endicott and Ling 1989). However, CQR isolates of P. falciparum do not display such striking cross-resistance as occurs in mammalian MDR. It is clear that acquisition of chloroquine resistance does not guarantee resistance to either quinine, amodiaquine, or mefloquine but it does appear to predispose the parasite to resistance to these other drugs. For example, a study in East Africa followed the appearance of chloroquine resistance and the data showed a gradual increase in quinine and amodiaquine resistance and some examples of mefloquine resistance despite the fact that

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these drugs had not been used in this area (Draper et al. 1988).

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perhaps the pfmdrl gene of CQR parasites was mutated at one or more positions within the primary sequence. The potential AMPLIFICATION OF mdr GENES functional alteration manifested at the level The MDR phenotype in mammalian tu- of the gene product might thus render a parmor cells is often associated with amplifi- asite competent for CQR. The presence of cation of mdr genes and overexpression of sequence differences in the pfmdrl gene the protein product. Foote and colleagues was later confirmed and enabled the accu(1989) first demonstrated amplification of rate prediction of the chloroquine resissegments of DNA on chromosome 5 con- tance or sensitivity status of a further 34 of taining the pfmdr2 gene in some but not all 36 isolates in a study based on the sequence CQR isolates of P. falciparum. More re- of distinct regions of pfmdrl alone (Foote et cently, a larger survey of CQR isolates has al. 1990). It is important to note that the shown that a relatively high proportion of majority of these isolates were not fully sethese isolates also have amplified genes quenced and that many of the isolates were (Triglia et al. submitted for publication). not cloned. These two factors may explain, Analysis of the breakpoints for amplifica- in part, the two apparent anomalies in this tion showed that most isolates possessed study which were incorrectly predicted. It different sized pfmdrl-containing ampli- also seems likely that other pfmdrZ alleles cons, leading to the suggestion that ampli- exist and that other mutations may also be fication of pfmdrl has arisen as a number of linked to CQR. The availability of multiple independent events. These results suggest allelic forms of pfmdrZ may provide a usethat the area of the genome containing the ful tool for the analysis of structure/ pfmdrl gene is under strong selective pres- function relationships at the level of the sure. The questions of particular interest corresponding Pgh. The presence of mutations in other memare, therefore, why does amplification of pfmdrl occur and is it in any way related to bers of the P-glycoprotein-like family may CQR? Although many CQR isolates pos- provide some insight into the functional sigsess only a single copy of pfmdrZ, there nificance of mutations in pfmdrl . In human P-gp, for example, a single mutation resultmust be selective pressure for amplification ing in a glycine to valine substitution at poof this region of the genome of P. falciparum. It is possible that amplification is sition 185 dramatically alters its substrate somehow related to the temperogeographic specificity (Choi et al. 1988). A valine at spread of CQR. Thus, did some isolates this position predisposes for preferential which presently contain a single copy of colchicine resistance while cells expressing pfmdr2 previously have multiple copies wild-type P-gp exhibit preferential resiswhich underwent deamplification as other tance to vinblastine. This alteration in the genetic changes occurred in response to rank order of drug resistance is apparently chloroquine pressure? While the signifi- reflected in a change not at an initial drug cance of pfmdrl gene amplification is not binding site but at a different site incorpounderstood, it is clear that amplification of rating the mutant residue which is more intimately involved in drug translocation pfmdrl is insufficient for CQR. (Safa et al. 1990). A related phenomenon MUTATIONS OF pfmdrl LINKED TO CQR has been observed in murine P-glycoproThe presence of the pfmdrl gene in some teins with mutations introduced by siteCQR isolates at a copy number equivalent directed mutagenesis (Gros et al. 1991). Acto that of chloroquine-sensitive (CQS) par- cordingly, a single amino acid substitution asites led Foote et al. (1989) to suggest that involving residue 939 profoundly modulates

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the drug resistance profiles of transfectant cell lines expressing P-glycoproteins bearing a mutant residue at this position. In the case of the mouse mdrl gene product, a mutation at position 939 results in a P-glycoprotein capable of conferring resistance to vinblastine but which has lost the ability to confer cross resistance to colchicine and adriamycin. Significantly, this mutation, which may be important in the underlying mechanism common to distinct mouse P-glycoproteins, maps to the homologous residue which is altered in one of the pfmdrl alleles that has been linked to CQR. Thus, phenotypic changes resulting in distinct specificity profiles can be altered by mutations in P-gp. Similarly, mutations are a common feature of allelic forms of the gene product which is defective in cystic fibrosis (CFTR). Mutant residues within this polypeptide, which is also a member of the P-gp-like superfamily, have been directly linked to the major defect of altered chloride permeability (Riordan et al. 1989; Rich et al. 1990; Kerem et al. 1990). It seems reasonable to suggest that mutations in pfmdrl may also lead to functional alterations. Whether these changes are in any way linked to the intracellular distribution of chloroquine has not been demonstrated. THE GENETIC

BASISOF CQR

CQR is known to have arisen from two foci: one in South America and one in Southeast Asia in the late 1950s and early 1960s (Maberti 1960; Harinasuta et al. 1962). The length of time intervening between the introduction of chloroquine on a wide scale and the first reported cases of resistance, as well as the time taken for resistance to spread despite widespread use, supports the notion that CQR is indeed a complex phenotype. In agreement with this is the fact that it has not been possible to select, in vitro, a CQR P. fulcipurum line from a CQS parent, again suggesting that multiple genetic events are required. Resistance to other drugs such as mefloquine,

however, can be readily selected once CQR has been established (Oduola et al. 1988), a feature which will undoubtedly affect the future of antimalarial drug use. It seems likely that the genetic basis of CQR involves alterations in more than one gene. This has been shown to be the case in P. chubuudi in which CQR segregates in a manner consistent with a multigenic phenotype (Rosario 1976). However, a single genetic cross performed by Wellems and colleagues (Wellems et al. 1990)suggeststhat the inheritance of CQR in P. fulcipurum behaves as a single genetic locus and that pfmdrl and pfmdr2 segregate independently of the CQR phenotype. Subsequent analysis of the progeny of this cross has localized a gene to a 400-kb region of chromosome 7 that is linked to the rapid efflux phenotype that characterizes chloroquine resistance (Wellems et al. 1991). This genetic analysis will allow the eventual isolation of a gene that appears to be important in this phenotype. The suggestion, from the genetic cross, that a single locus independent of pfmdr genes is involved in the rapid efflux phenotype is in contrast with the report that mutations in pfmdrl are strongly linked to the CQR phenotype. While the interpretations of the genetic basis of CQR suggested by these two groups differed, the data could be reconciled on the basis of mutations in pfmdrl (Foote et al. 1990). The advent of an efficient P. fulcipurum transfection system should ultimately allow this matter to be resolved. EXPRESSION AND SUBCELLULAR LOCALIZATION OF THE pfmdrZ GENEPRODUCT

It has recently been shown that the gene product of pfmdrl is a polypeptide of approximately 160,000 Da consistent with the molecular weight predicted from the nucleotide sequence and has been called Pghl (Cowman et al. 1991). Analysis of this protein in a number of P. fulcipurum isolates has revealed that all parasite lines regard-

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less of their chloroquine resistance or sensitivity status express Pghl. The polypeptide is expressed throughout the asexual erythrocytic cycle and therefore is present (particularly in the trophozoite stage) when chloroquine exerts its antimalarial activity. However, many CQR isolates express about the same level of Pghl as do their CQS counterparts. For example, the Kl CQR cloned isolate contains a single copy of the pfmdrl gene and expresses equivalent amounts of Pghl as all CQS isolates that have been analyzed. The CQR cloned line, FACS, which has three copies of the pfmdrl gene expresses approximately threefold more Pghl than other CQR isolates (Cowman et al. 1991). Since FAC8 was not more resistant to chloroquine than other CQR lines expressing lower levels of Pghl, there is no correlation between the level of CQR and the amount of Pghl expressed. This suggests that Pghl may not be acting as a direct efflux pump for chloroquine but does not rule out the possibility that it is involved in a more indirect way. Using antibodies raised against the pfmdrl gene product, it was possible to localize Pghl to the membrane of the digestive vacuole of trophozoites (Cowman et al. 1991). This localization on the membrane of the chloroquine accumulating compartment has important implications for its function and a potential role in the CQR phenotype. Since Pghl has retained the basic structural motifs preserved throughout evolution in other prokaryotic and eukaryotic transport systems, it seems likely that it is involved in a nucleotide-dependent transport function across the food vacuole membrane. Although the substrates for Pghl are not known, it appears as though the polypeptide is uniquely positioned to influence intravacuolar CQ concentrations either directly or indirectly. Thus, the function of mutant Pghl present in CQR P. falciparum may in some way be linked to the major phenotypic feature of CQR: the lack of significant intravacuolar CQ accumulation in

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the resistant versus the sensitive parasite. Moreover, the location of Pghl also suggests a mechanism for the enhancement of chloroquine action by chemosensitizing drugs. Accordingly, these compounds may inhibit the function of Pghl leading to a situation in which toxic levels of chloroquine might be accumulated in the digestive vacuoles of CQR parasites, a suggestion which is based on the known interaction of reversal agents with mammalian P-gps. THE

MECHANISM

CHEMOSENSITIZING

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DRUGS

In mammalian MDR tumor cells, it has been shown that expression of P-gp is sufficient to confer the drug resistance phenotype (Guild et al. 1988) and that the level of plasma-membrane-associated P-gp approximates the level of drug resistance observed (see Endicott and Ling 1989). The recent studies on the genetic basis of CQR (Wellems et al. 1990, 1991) and the demonstration of mutations in pfmdrl linked to CQR (Foote et al. 1990) suggest that expression of mutant forms of Pghl alone is insufficient for CQR. Similarly, analysis of the expression and subcellular localization of Pghl in P. falciparum also supports the notion that overexpression of Pghl on the digestive vacuole membrane is not correlated with the level of CQR (Cowman et al. 1991). Therefore, it is possible that Pghl may not function in a manner analogous to the mammalian P-gps which are involved in drug resistance. What then is responsible for the common phenotypic features of CQR and MDR? Furthermore, what is the molecular basis of the action of chemosensitizing drugs in CQR isolates of P. falciparum? One hypothesis which has been put forward and supported experimentally is the pH gradient hypothesis (Geary et al. 1986, 1990). It is generally accepted that the pH gradient between the medium and the parasite digestive vacuole is the driving force for accumulation of chloroquine. It has

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been shown that the intravacuolar pH of several CQR isolates is somewhat higher than that of CQS isolates and that this might account for the observation that chloroquine does not accumulate significantly within the digestive vacuoles of CQR parasites (Geary et al. 1990). How these parasites tolerate the change in intravacuolar pH or how the alteration of pH is accomplished is as yet unknown. Two predictions that follow from this model are first, that the so-called reversal agents might function by decreasing the pH of the relatively more basic food vacuole compartment of CQR parasites, and secondly, that the true crossresistance might be expected to occur in the case of the quinoline-containing antimalarials. The idea of altered intravacuolar pH is interesting in light of recent observations that P-glycoprotein activity results in alkalinization of the drug-accumulating compartment in mammalian MDR tumor cells (Thiebaut et al. 1990; Keizer and Joenje 1989). The possibility exists therefore that the P. falciparum Pghl may in some way be linked to the regulation of intravacuolar pH. The mechanism of action of the family of structurally unrelated reversal drugs capable of sensitizing CQR P. falciparum to chloroquine is also of some interest with the respect of Pghl. It has been suggested that the site of action of these compounds is the food vacuole since their effect can be abrogated by raising the intravacuolar pH (Krogstad et al. 1987). Although the intracellular target of chloroquine is still not defined, it is possible that chemosensitizing drugs enhance the action of chloroquine at the level of the target. It has been shown, however, that these compounds increase the retention of chloroquine by CQR parasites and may act by enhancing the intrinsic antimalarial properties of chloroquine (Krogstad er al. 1987; Kyle et al. 1990). Because of its localization and the circumstantial evidence linking Pghl to the CQR phenotype, Pghl is a candidate as a target for reversal drugs.

There are many similarities between the MDR and CQR phenotypes of mammalian tumor cells and P. falciparum but there are also some differences. It is possible that the underlying mechanisms involve similar effector molecules, but despite strong circumstantial evidence linking the protein product of the pfmdrl gene to CQR, there is no direct functional proof. While genetic evidence suggests that the mdr homologues of P. fulciparum are not directly involved in this phenotype (Wellems et al. 1990) the data can be explained if a second gene is essential (Foote et al. 1990). The genetic locus identified on chromosome 7 appears to be an important component involved in the rapid efflux phenotype of CQR (Wellems et al. 1991). The isolation of this genetic locus will hopefully shed light on this important and complex problem. ACKNOWLEDGMENTS We thank David Kemp for reviewing the manuscript. Work that has taken place at The Walter and Eliza Hall Institute of Medical Research was supported by grants from the National Health and Medical Research Council of Australia and the John D. and Catherine T. MacArthur Foundation. S.K. is supported by a postdoctoral fellowship from the Canadian Medical Research Council. The support of a Wellcome Australian Senior Research Fellowship in Medical Science for AFC is also acknowledged. REFERENCES BECK, W. T., CIRTAIN, M. C., LOOK, A. T., AND ASHMUN, R. A. 1986. Reversal of vinca alkaloid resistance but not multiple drug resistance in human leukemic cells by verapamil. Cancer Research 46, 788-794. BITONTI, A. J., SJOERDSMA,A., MCCANN, P. P., KYLE, D. E., ODUOLA, A. M., ROSSAN, R. N., MILHOUS, W. K., AND DAVIDSON, D. E. J. 1988. Reversal of chloroquine resistance in the malaria parasite Plasmodium falciparum by desipramine. Science 242, 1301-1303. BRUGGEMANN, E. P., GERMANN, U. A., GOTTESMANN, M. M., AND PASTAN, I. 1989. Two different regions of P-glycoprotein are photoaftinity-labelled by azidopine. Journal of Biological Chemistry 264, 15,483-15,488. CANO-GAUCI,D. F., BUSCHE,R., TUMMLER, B., AND RIORDAN, J. R. 1990. Fast kinetic analysis of drug transport in multidrug resistant cells using a pulsed

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Received 8 May 1991; accepted 10 May 1991