active efflux by multidrug transporters as one of the strategies

Pharmucol. T/W. Vol. 76,Nos. 1-3,~~. 219-242, 1997 Copyright 0 1997 Elsevier Science Inc.

ISSN0163-7258/97$32.00 PI1 SO163-7258(97)00094-6

ELSEVIER

Associate Editor: D. Shugar

Active Efflux by Multidrug Transporters as One of the Strategies to Evade Chemotherapy and Novel Practical Implications of

Yeast Pleiotropic Drug Resistance

Marcin Kolaczkowski and Andre’ Goffeau” UNIT~DEBlOCHlMIEPHYSIOLOGlQUE,UNIVERSIT~CATHOLIQUE DELOUVAIN,PLACECROlXDUSUD2/20,

B-1348 LOUVAIN LA NEUVE, BELGIUM

ABSTRACT. Mankind is faced by the increasing emergence of resistant pathogens, including cancer cells. An overview of the different strategies adopted by a variety of cells to evade chemotherapy is presented, with a focus on the mechanisms of multidrug transport. In particular, we analyze the yeast network for pleiotropic drug resistance and assess the potentiality of this system for further understanding of the mechanism of broad specificity and for development of novel practical applications. PHARMACOL. THER. 76:( l-3):219-242, 1997. 0 1997 Elsevier Science Inc.

KEY WORDS. Chemotherapy, antibiotic, fungicide, anticancer. substrate specificity.

CONTENTS

1. INTRODUCTION . . . . . . . . . . . . . 219 2. TRICKSTOSURVIVE . . . . . . . . . . . 220

2.1. 2.2. 2.3.

2.4. 2.5.

2.6.

2.7.

ENZYMATICINACTIVATION . . . . 220 TARGETALTERATIONS. . . . . . . 222 ALTERATIONSOFACTIVITYOF DRUGTARGET-AND DRUG-METABOLIZINGENZYMES . . 222 INCREASEDDNAREPAIR . . . . . 223 FAILURETOUNDERGO APOPTOSIS . . . . . . . . . . . . . 223 REDUCEDACCUMULATIONAND SEQUESTRATION . . . . . . . . . . 223 AcTIvEEF~ux........... 224 2.7.1. CANCERCELLS . . . . . . . 224 2.7.2. OTHERPATHOGENS. . . . . 225

3. THEDILEMMAOFBROADSUBSTRATE SPECIFICITY . . . . . . . . . . . . . . . 227 3.1. DRUG-RESISTANCEPROFILES

3.2. 3.3.

OFMULTIDRUG-RESISTANCE TRANSPORTERS . . . . . . . . . . . 227 MECHANISM. . . . . . . . . . . . . 229 SUBSTRATERECOGNITION BYMULTIDRUG RESISTANCE-ASSOCIATED PROTEIN............... 230

4. THEYEASTSACCHAROMYCES CERIWISIAE fiEIOTROPIC DRUG-RESISTANCENETWORK ASATOOLTOUNDERSTANDTHE MECHANISMOFMULTIDRUG RESISTANCEANDTRANSPORT . . . . . 231

5. OTHERPRACTICALIMPLICATIONSOF THEYEASTPLEIOTROPIC DRUG-RESISTANCENETWORK . . . . . 232

ACKNOWLEDGEMENTS............ 234 REFERENCES . . . . . . . . . . . . . . . . . 234

ABBREVIATIONS. ABC, ATP-binding cassette; AIDS, acquired immunodeficiency syndrome; AM, ace- toxymethyl; Al’, abasic (a purinic or a pyrimidinic); ARA, anthracycline resistance-associated; CFTR, cystic fibrosis transmembrane conductance regulator; DNP-SG, dinitrophenol-S-glutathione; LRP, lung resistance- related protein; LT, leukotriene; MDR, multidrug resistance; MFS, major facilitator superfamily; MK57 1, 3( [(3(2[7-chloro-2-quinolinyl]ethenyl)phenyl} ((3-dimethylamino-3-oxopropyl) thio] methyl] thio) propanoic acid; MRP, multidrug resistance-associated protein; NBD, nucleotide-binding domain; 4NQO,+nitroquinoline- N-oxide; PDR, pleiotropic drug resistance; P-gp, P-glycoprotein; RND, resistance/nodulation/cell division; SMR, small multidrug resistance; Texans, toxin-extruding antiporters; TM, transmembrane; TMA-DPH, l-[+(trimethylamino) phenyll-6-phenylhexa-1,3,5triene.

1. INTRODUCTION

An immense variety of small organic molecules was de-

signed in the course of evolution as toxic weapons of chem-

ical warfare carried on by microorganisms in their fight for

survival and elimination of competitors. They are also abundant in plants as a means of killing or repelling herbi- vores and pathogenic microbes or other plants (Taiz and Zeiger, 1991). Moreover, to protect themselves against in-

*Corresponding author.

fections, plants, as well as animals and humans, have developed a defense system based on broad-spectrum hydrophobic antimicrobial peptides. In vertebrates, these supplement

cell-mediated immunity, protecting mucosal surfaces of the respiratory and gastrointestinal tracts, as well as skin, against invading microorganisms (Nicolas and Mor, 1995).

To balance this toxic arsenal, which has been extended during the last 50 years by the development and massive use of antibiotics, evolution has equipped living organisms with a plethora of protecting systems. These systems, to our annoyance, contribute to the large-scale emergence of resis-

220 M. Kolaczkowski and A. Goffeau

tant pathogens, as stressed in many recent alarming reports (Neu, 1992; Krause, 1992; Travis, 1994; Gold and Moeller- ing, 1996; Acar and Goldstein, 1997). These include vancomycin-resistant enterococcal bacteremia, for which there is no effective antibiotic treatment (Morris, J. G. et al., 1995; Linden et al., 1996), multidrug-resistant tuberculosis (Morris, S. et al., 1995; Huebner and Castro, 1995; Kaye and Frieden, 1996; Blanchard, 1996), Plasmodium spp. (Borst and Ouelette, 199.5; Rubio and Cowman, 1996; But- ler, 1997) and other parasitic protozoa (Borst and Ouelette, 1995; Ullman, 1995; Rubio and Cowman, 1996), as well as resistant helminths (Geerts et al., 1997). The number of drug-resistant fungal clinical isolates also tends to increase (Odds, 1996). The chemotherapeutic treatments used in the global acquired immunodeficiency syndrome (AIDS) pandemic, as well as the use of drugs inducing severe immu- nosuppression in transplant and cancer patients, additionally contribute to the incidence of new and drug-resistant pathogens (Huebner and Castro, 1995; Hazen, 1995; Hazen, 1995; Odds, 1996; Kelly et al., 1997).

Although endogenous defense mechanisms protect us from natural toxins, such as the plant pesticides phenols, flavonoids, alkaloids, glucosinolates and saponins, con- sumed daily in food, and many others, including, for example, the potent mold carcinogens aflatoxins, they can also sometimes turn against us. Thus, tumors originating from cells lining excretory, detoxifying organs, such as the kidney, gut and liver, are often intrinsically resistant to anticancer drugs. Many other tumors acquire resistance during the course of chemotherapy (Mattem and Volm, 1993).

One of the important and clinically relevant mechanisms of drug resistance in human malignancies is the active extrusion of toxic chemicals by broad specificity multidrug resistance (MDR) transporters (Gottesman and Pastan, 1993, 1997; Mattern and Volm, 1993; Simon and Schindler, 1994; Skovsgaard et al., 1994; Gottesman et al., 1995; Kane, 1996; Borst and Schinkel, 1997). The potential danger of this type of mechanism, widespread among bacterial, fungal and parasitic pathogens, lies in the fact that a single protein can confer resistance to a plethora of compounds from different chemical classes. This broad specificity is in clear contrast to many other types of resistance, which are usually limited to a single drug or to a class of structurally closely related drugs. Spontaneous mutations yielding even more effective transporters for selective agents or transporters with altered specificity profiles have been reported (Choi et al., 1989; Chen et al., 1997; Kly- achko, et al., 1997). Transporters encoding genes can be spread by means of plasmids and transposons (Roberts, 1994), as has happened with many other drug-resistance genes (Davies, 1994). Indeed, some MDR transporters, such as QacA and Smr, conferring resistance to disinfectants and antiseptics, already have been identified on plasmids from clinical isolates of staphylococci (Littlejohn et al., 1992; Leelapom et al., 1994).

To overcome MDR transporters, the understanding of their mechanism of action and regulation is of key clinical

and agricultural importance. In the long term, it should allow manipulation of the specificity profile in a controlled and predictable way to tailor it for our benefit. We believe that yeast has much to offer to the study of MDR and will describe below recent developments and new prospects in this field.

Before going into more detail on MDR transporters, we will present a brief overview of other types of resistance mechanisms, since, in most cases, the resistance is multifac- torial and different mechanisms often link together to cause a poor response to chemotherapy.

2. TRICKS TO SURVIVE

Various resistance mechanisms found in many microbial and parasitic pathogens and cancers are often present in different supplementing combinations and include: (1) inactivation of drugs by enzymes, (2) target alterations resulting in decreased target affinity, (3) changes in the cellular activity of target enzymes or drug-activating enzymes, (4) increased DNA repair, (5) failure to undergo apoptosis by cancer cells, (6) reduced accumulation and sequestration, and (7) active efflux (Fig. 1.).

2.1. En-tic Inactivation

Enzymatic inactivation is the major mechanism of resistance toward P-lactam antibiotics and results from hydroly sis of the p-lactam ring of penicillins, cephalosporins and related antibiotics ‘(Livermore, 1995). Resistance to newly generated p-lactams due to point mutations in p-lactamases is common (Davies, 1994; Gold and Moellering, 1996).

Enzymatic modification is also the most clinically signifi- cant mechanism of inactivation of aminoglycosides (streptomy tin, kanamycin, gentamycin, neomycins) by aminoglycoside acetyltransferases, phosphotransferases, nucleotidyltransferases and adenylyltransferases, often associated with plasmids or transposons (Shaw et al., 1993). Here, in contrast to p-lactamases, specificity alterations of modifying enzymes have not been identified in clinical isolates (Davies, 1994).

Also widely distributed among bacterial pathogens are chloramphenicol acetyltransferases (Shaw and Leslie, 1989). Many macrolide-, lincosamide- and streptogramin-inactivating enzymes (acetyltransferases, hydrolases, esterases, nucleotidyltransferases, phosphotransferases, and glycosyl transferases) have been detected in clinical isolates, although they are less important clinically than p-lactamases (Arthur et al.,

1987; Kono et al., 1992). Fosfomycin, used in the treatment of sepsis, is inacti-

vated by its derivatization with glutathione, by the plasmid- encoded glutathione S-transferase (Suarez and Mendoza, 1991). This is the only example of this type of mechanism in bacteria, which is widespread in other organisms.

In fungi, plants, animals, and humans, the intracellular metabolism of endo- and xenobiotics is mediated by cytochrome P450 monooxygenases (Kivisto et al., 1995) and further by glutathione S-transferases (Commandeur et al.,

Drug Resistance Mechanisms 221

222

1995; Hayes and Pulford, 1995), UDP-glucuronyl transferases (Bock, 199 1) or sulfotransferases (Falany, 199 1) . These conjugations are key defense mechanisms in the detoxification of endogenous and exogenous electrophilic- reactive compounds in eukaryotes. Not only do they eliminate harmful electrophilic moieties, but they provide a mo-

lecular “flag” that signals export of the conjugate from the cell. This mechanism contributes to resistance to chemo-

therapy of cancer-cells.

Various types of other enzymatic inactivation mecha-

nisms have been observed in fungi, plants, animals, and humans. Bleomycin hydrolase, for example, is a cysteine pro-

tease involved in tumor resistance to the anticancer drug

bleomycin. Increased mRNA levels for this hydrolase have been observed in a variety of tumor cell lines (Bromme et

al., 1996).

2.2. Target Alterations

Development of targets with reduced affinity for antibiotics is a major mechanism of resistance when drug-inactivating

or -modifying enzymes are absent. It occurs most rapidly

and frequently with drugs that inactivate a single target and are not substrate analogs. Most frequently, it results from

target modifications, mutations, or acquisition of drug- insensitive enzymes replacing the target function.

Methylation of ribosomal RNA by rRNA methylases, of-

ten carried on plasmids and transposons, is the most often encountered mechanism of bacterial resistance to macrolides, lincosamides and streptogramins in clinical isolates

(Leclerq and Courvalin, 1991). Resistance to tetracyclines, often encoded by transposons and conjugative plasmids

(Roberts, 1994), can be mediated by protection of the ribo- some by TetO or TetM, whose functions at the molecular

level have not been fully elucidated yet (Schnappinger and

Hillen, 1996). Mutations in the drug targets, resulting in decreased drug

binding, occur for all types of drugs. For example, mutations in DNA gyrase confer resistance to fluoroquinolones in bacteria, including Sta@ylococcus aureuS (Ouabdesselam et

al., 1996). Therapeutic options for Staphylococcus aurew are quite limited, especially in the case of methicillin-resistant strains. Resistance to p-lactam antibiotics can result from

alterations in the penicillin-binding proteins (transpepti- dases participating in bacterial cell wall biosynthesis), by creation of hybrid proteins through interspecies recombina- tion (Spratt, 1994). Mutations in topoisomerases I and II

are one of the mechanisms of resistance to anticancer drugs targeted to topoisomerase I (camptothecin, topotecan) and II (etoposide, mitoxantrone, doxorubicin, amsacrine, ellip- ticine, saintopin) (Nitiss and Beck, 1996). Resistance to azole fungicides in clinical isolates can result from their decreased affinity for the target enzyme, the cytochrome P450-dependent sterol 14-cY-demethylase (Marichal and Vanden Bossche, 1995 ) .

Combination therapy with drugs affecting different targets strongly diminishes the chance of simultaneous resis-

M. Kolaczkowski and A. Goffeau

tance development (Kremsner et al., 1997). Accumulation of mutations in the individual drug targets is the most frequent mechanism of MDR to escape antimycobacterial drugs such as streptomycin, rifampin and isoniazid (Morris,

S. et al., 1995; Blanchard, 1996). Multiple resistance to nucleoside analogues and non-nucleoside human immunodeficiency virus reverse transcriptase inhibitors, due to acquisition and accumulation of point mutations in the target

enzyme, is also frequent during AIDS chemotherapy, where the high proviral DNA transcription replication rates and

the resulting variability contribute to its quick develop-

ment (Schmit et al., 1996; Brown and Richman, 1997). A

similar mechanism of resistance of the human immunodeficiency virus protease to its potent inhibitors, indinavir, ritonavir and others, has also been reported (Molla et al.,

1996; Brown and Richman, 1997). Acquisition of new target enzymes (dihydropteroate syn-

thase and dihydrofolate reductase) with reduced affinity for the inhibitor is the major mechanism of resistance to sul-

fonamides and trimethoprim in bacteria (Huovinen et al.,

1995). Acquisition of a transposon-encoded (additional) penicillin-binding protein with low affinity for p-lactam

antibiotics, which takes over the function of endogenous enzymes, is the mechanism of methicillin resistance in St&-

ylococcus aureus (Song ec al., 1987). Until recently, vancomycin was the only reliable drug to treat infections caused

by multidrug-resistant enterococci. Vancomycin-resistant

microbes have spread recently in hospitals (Morris, J. G. et al., 1995; Linden et al., 1996). Acquired vancomycin resistance results from acquisition of the plasmid-encoded op- eron mediating the synthesis of altered peptidoglycan precursors of reduced affinity for the antibiotic (Walsh, 1993).

2.3. Alterations of Activity of Drug Target- and Drug-Metabolizing Enzymes

Resistance can also arise by increased target enzyme activity. For example, resistance towards the anticancer drug methotrexate often results from overproduction of dihydro-

folate reductase by amplification of its gene, but sometimes from increased mRNA translation (Skovsgaard et al.,

1994). The opposite is observed with topoisomerases I and II,

which provide the strand breakage-unwinding-ligation ac- tivities crucial for DNA replication. Their inhibitors stabi-

lize the DNA-topoisomerase complexes, leading to formation of double-strand breaks and cell death. Resistance to topoisomerase inhibitors, due to decreased enzyme levels, or reduced activity by mutations in the ATP-binding domain, have been observed (Nitiss and Beck, 1996; Houl-

brook et al., 1996). Many drugs must be metabolized inside the cell to their

active form, and any changes in this process can lead to resistance. The antifungal 5-fluorocytosine exerts its cytotoxic effect upon intracellular conversion and inhibition of DNA synthesis, as well as incorporation into RNA, which is then aberrant. Upon entry to the cell through cytosine


permease, it is deaminated by cytosine deaminase to ~&IO- rouracil, which is then converted by uridine monophosphate pyrophosphorylase into 5-fluorouridylic acid. This is further phosphorylated and incorporated into aberrant RNA. 5Fluorouracil is also converted simultaneously into 5-fluorodeoxyuridine monophosphate, a potent inhibitor of thymidylate synthase. Resistance to 5-fluorocytosine can result from loss or mutation of any of the enzymes involved in its activation, and is so common that this antifungal is no longer recommended for single-drug therapy (Vanden Bossche et al., 1994).

2.4. Increased DNA Repair

DNA is the site of action of many anticancer agents that can bind covalently (nitrogen mustards and nitrosoureas). Some bifunctional agents produce intra- or interstrand crosslinks (cisplatin, nitrogen mustards). Others bind non- covalently, by intercalation between base pairs (anthracy- clines) or by nonintercalative groove binding (distamycin A). If the extent of DNA damage is high, cells die. Other- wise, small lesions can be repaired by three main mechanisms: (1) direct repair, in which the chemical bonds link- ing the substituent to DNA are cleaved; (2) base excision, in which the modified base is first removed by a glycosylase, followed by removal of the abasic (AP) site by AP lyase/en- donuclease, with subsequent nucleotide replacement; (3) nucleotide excision repair, making nicks on both sides of the damaged region, which is then released and filled in (Sancar, 1995). Increased DNA repair is often associated with resistance to DNA-damaging anticancer agents (Skovs- gaard et al., 1994; Zamble and Lippard, 1995; Sancar, 1995).

2.5. Failure to Undergo Apoptosis

The primary mechanism by which most anticancer drugs exert their cytotoxic effects is the creation of disturbances in cellular metabolism, leading to induction of apoptosis (Seimiya et al., 1997). Apoptosis is the tightly regulated, intrinsic cellular suicide program that assures homeostasis in multicellular organisms, the complexity of which we are only now beginning to understand (Steller, 1995; Ander- son, 1997; McCall and Steller, 1997). Cancer cells escape this natural regulatory mechanism and proliferate in an un- controlled way. They often show decreased ability to undergo apoptosis in response to at least some physiological signals; when these include signals elicited by anticancer drugs, resistance to chemotherapy results (Thompson, 1995; Mashima et al., 1996; Reed et al., 1996; Hannun, 1997).

2.6. Reduced Accumulation and Sequestration

Although reduced cellular accumulation of drugs is often a consequence of increased efflux, we will focus first on other mechanisms.

Many hydrophilic drugs, for example, the anticancer an- timetabolite methotrexate, cannot easily diffuse through the

plasma membrane and have to use specific transporters for this purpose. Alterations in these transporters often lead to

reduced influx of drugs. In the case of methotrexate, resistance was related to alterations in the folate transporter (Skovsgaard et al., 1994), a mechanism common in other species.

Many gram-negative microorganisms, such as Pseudomo- nas spp., are intrinsically resistant to many antibiotics due to extremely low permeability of their outer membrane (Nakae, 1995), which is composed of lipopolysaccharide units of several tightly packed unsaturated hydrocarbon chains linked to a single polar head group. The nutrients enter through porin channels, which, due to their small pore size, exclude many antibiotics (Nikaido, 1994). Re- duced production or mutational alterations of porins in gram-negative bacteria contribute to their acquired resistance to some antibiotics (Martinez et al., 1996). An ex- treme case are mycobacteria, which are intrinsically resistant to most antibiotics unable to penetrate through the thick and highly ordered outer membrane containing chains of mycolic acid more than 70 carbons long (Brennan and Nikaido, 1995).

Decreased toxicity can also be caused by changes in the lipid composition of the membranes, leading to a decrease in permeability. This is the mechanism of bacterial resistance to organic solvents, some of which are used as disinfectants. The decreased permeability and fluidity of the membranes results from cis- to tram-isomerisation of their unsaturated fatty acids (Heipieper et al., 1994). Resistance to the polyene systemic antifungals nystatin and amphotericin B, which interact with ergosterol to form pores in membranes (Cohen, 1992), results, in most cases, from de- fects in the ergosterol biosynthesis pathway, leading to a decreased ergosterol level in the plasma membrane (Kelly et al., 1997). Existence of other mechanisms of polyene resistance, not linked to sterol alterations, has also been suggested (Josephhorne et al., 1996).

The reduction of membrane permeability due to changes in membrane biophysical properties, or loss of porins, is not a very efficient way of resistance, unless it is accompanied by another resistance mechanism, such as active efflux or enzymatic inactivation, which is often the case (Nikaido, 1994; Thanassi et al., 1995).

As in the case of solid tumors, formation of cell clusters imposes another kind of permeability barrier. These large masses of cells are usually poorly vascularized, reducing their penetration by anticancer drugs. This also causes limited oxygen and nutrient supply to subpopulations of cells, conditions that are linked to increased expression of drug- resistance genes (Simon and Schindler, 1994; Koomagi et al., 1995).

Yet another way of preventing access of a drug to its target is sequestration by binding to serum proteins. Drugs can also be sequestered in subcellular organelles by active transport, as for vesicular monoamine transporters (Yelin and Schuldiner, 1995; Schuldiner et al., 1995), or by membrane potential (A?) or pH gradient (ApH)-driven diffusion into


acidic organelles such as lysosomes or vacuoles. Daunorubi- tin, an anticancer drug, has been shown to accumulate in acidic organelles in ho; this occurs to a higher extent in some multidrug-resistant cells (Hindenburg et al., 1989). In many

tumor cell lines, the development of MDR has been correlated with an increase in intracellular pH, leading to de-

creased drug accumulation (Simon and Schindler, 1994). Accumulation of many chemotherapeutic compounds in li-

posomes, in response to membrane potential (A*) and a pH gradient ( ApH), has been well documented and includes the anticancer drugs vinblastine, vincristine, doxorubicin, daunorubicin, epirubicin, and mitoxanthrone; the

antimalarials chloroquine and quinine; and many others (Cullis et al., 1991). Interestingly, the observations of Wei

et al. (1995) show that over-expression of the cystic fibrosis transmembrane conductance regulator (CFTR) lowers the

membrane potential and intracellular pH and confers resistance to anticancer drugs such as doxorubicin, vincristine,

and colchicine.

2.7. Active EffEwr

Active efflux by specific or MDR transporters is a strategy

to prevent the access of toxic compounds to their intracellular targets. It is also used to excrete products of intra-

cellular metabolism of endo- and xenobiotics, “flagged” by glutathione, glucuronide, or sulfate. Drug transporters,

widespread from bacteria to humans, are found within four families of proteins. These are the ATP-driven ATP-bind-

ing cassette (ABC) transporter superfamily (Doige and

Ames, 1993; Fath and Kolter, 1993); the proton motive force-driven toxin-extruding antiporters (Texans), a subgroup within the major facilitator superfamily (MFS); the small multidrug resistance (SMR) family, or miniTexans; and the resistance/nodulation/cell division (RND) family

(Paulsen et al., 1996a,b; Goffeau et al., 1997; Schuldiner et al., 1997).

Other members of the ABC transporter superfamily, not shown to be involved in drug resistance, are involved in se-

cretion, uptake, or intracellular transport of a large variety

of different substrates, including proteins, peptides (Fath and Kolter, 1993; Kuchler et al., 1994), lipids (Oude Elf-

erink et al., 1997) and different nutrients (Doige and Ames, 1993). Uptake of nutrients is also mediated by MFS transporters unrelated to drug resistance (Nelissen et al., 1995).

2.7.1. Cancer cells. In cancer cells, resistance to chemotherapy often results from active efflux mediated by the over-expressed MDRl P-glycoprotein (P-gp), a member of the ABC superfamily (Gottesman and Pastan, 1993, 1997; Gottesman et al., 1995; Kane, 1996). Multidrug-transporting P-gps, homologues of human MDRl, have also been identified in rodents. These comprise the mouse Mdrla and Mdrlb, rat Mdrla and Mdrlb, and hamster Pgpl and Pgp2 (Borst and Schinkel, 1997). P-gps extrude with different, but overlapping, specificities a large variety of toxic hydrophobic molecules in an unmodified form, utilizing energy

from ATP hydrolysis (Senior et al., 1995). P-gps are api- tally localized in epithelial cells lining the intestine and kidney, in the canalicular membrane of liver cells, in en- dothelial cells of the blood-brain barrier and in pluripotent

precursor stem cells of the bone marrow. These observations, and studies with mdrl/mdr2 knockout mice, which

did not develop any detectable phenotype apart from marked drug hypersensitivity (Borst and Schinkel, 1997),

suggest that these proteins play a key role in excretion and formation of an active permeability barrier for the toxic hydrophobic compounds, which are normal constituents of the environment and which otherwise easily diffuse through lipid bilayers (Eytan et al., 199613). The importance

of P-gp in the clinical resistance of several types of cancers

to antineoplastic drugs has been well established (Arceci, 1993; Filipits et al., 1996). The clinical relevance of differ-

ent drug-resistance genes in cancer has also been reviewed recently by Filipits et al. (1996).

A second identified human protein involved in clinically relevant broad specificity efflux of anticancer drugs and glu-

tathione conjugates is the MDR-associated protein (MRP), which is a homologue of MDRl and also belongs to the ABC superfamily (Cole et al., 1992; Lautier et al., 1996).

The activity of MRP strongly resembles that of the ATP- dependent GS-X pump, previously characterized biochemi-

cally. The GS-X pump excretes glutathione S-conjugates, cysteinyl leukotrienes (LTs), and certain organic ions from normal and cancer cells (lshikawa, 1992). It is considered

to modulate the resistance of cancer cells to cisplatin [cis-

diamminedichloroplatinum(ll)] (lshikawa et al., 1994).

The functional similarity, and the recent observations of

lshikawa et al. (1994, 1996), suggest that the GS-X pump and MRP are identical. It is unclear how many other pro-

teins are involved in transport of the previously discussed glutathione, glucuronide, or sulfate conjugates of endo- and xenobiotics, which are excreted mainly by liver and kidney,

and what is their contribution to cancer chemotherapy resistance. Some of these have been characterized biochemi-

cally, although the corresponding human genes have not

been identified yet (Commandeur et al., 1995). These include the dinitrophenol-S-glutathione (DNP-SG) ATPase, which is distinct from MRP and P-gp. DNP-SG ATPase transports glutathione S-conjugates and the anticancer

drugs doxorubicin, daunorubicin, and vinblastine (Awas- thi, Y. C. et al., 1992; Awasthi, S. et al., 1994; Saxena et al., 1992). Recently, the gene encoding a rat homologue of MRP, called cMoat, cMrp, or Mrp2, has been cloned (Pau- lusma et al., 1996). In contrast to MRP, which is expressed in several types of epithelia, muscle cells and macrophages

and is located basolaterally in liver cells, cMoat is expressed in the liver and is localized in the canalicular membrane, showing that although functionally related, these are two different proteins (Cole et al., 1992; Paulusma et al., 1996; Lautier et al., 1996).

Multidrug-resistant cancer cells often over-express the lung resistance-related protein (LRP), whose gene was cloned recently. The amino acid sequence of human LRP is


88% identical with the rat major vault protein. As vaults

are multisubunit structures involved in nucleocytoplasmic transport, it is likely that LRP mediates resistance by a

transport mechanism (Scheffer et al., 1995). Expression of LRP has been observed in many cancer cell lines, often together with P-gp and MRP (Izquierdo et al., 1996a,b), and is associated with a poor response to standard chemotherapy and adverse prognoses in patients with advanced ovarian carcinoma (Izquierdo et al., 1995) and acute myeloid

leukemia (List et al., 1996). There are reports suggesting the presence of an altema-

tive, other than the P-gp and MRP, efflux system for

daunorubicin in acute myeloid leukemia human cell lines (Hedley et al., 1997). A gene encoding a new ABC, anthra-

cycline resistance-associated (ARA) transporter, overexpressed in the anthracycline-selected human multidrug-resistant leukemia cell line together with MRP, has been cloned recently. ARA is the half-size transporter composed of six transmembrane (TM) spans and one nucleotide-binding domain (NBD) at the C-terminus. This is in

contrast to P-gps and MRP, in which this structure is doubled (TM-NBD-TM-NBD), with both halves being homol-

ogous. ARA is most similar to the C-terminal part of MRP (Longhurst et al., 1996). It is not known yet whether ARA

is involved in anthracycline efflux. Multidrug transport is also a feature of the previously

mentioned mammalian vesicular monoamine transporters,

which sequester drugs and neurotransmitters within synap- tic vesicles and other subcellular organelles and belong to the family of Texans; but their involvement in clinical cancer resistance has not been documented (Yelin and Schul- diner, 1995; Schuldiner et al., 1995).

2.7.2. Other pathogens. Active efflux of antibacterial,

antiparasitic, or antifungal agents has not been considered among the most prominent mechanisms of resistance until

recently. It began to attract attention following the discov-

ery of the plasmid-encoded energy-dependent efflux system for tetracycline (McMurry et al., 1980). More recently, awareness of the role of efflux mechanisms in microbial resistance has increased significantly (Nikaido, 1994; Poole, 1994; Williams, 1996; Jenkinson, 1996).

2.7.2.1. Bacteria. In bacteria, specific efflux systems contribute to clinical resistance to tetracycline, macrolides, and chloramphenicol. Active efflux of tetracyclines is usu-

ally mediated by proton motive force-driven transporters of the MFS, often carried on plasmids and transposons (Rob- erts, 1994). TetA, encoded by transposon TnlO, is the best

known (Schnappinger and Hillen, 1996). Resistance to macrolides in some clinical isolates of St&ylococcus aureus has also been associated with efflux. The identified MsrA transporter belongs to the ABC superfamily (Ross et al., 1990). A plasmid-encoded efflux mechanism, conferring resistance to 14- and 15-membered macrolides, has been isolated from Smphylococcus epidermidis (Goldman and Cap- pobianco, 1990). Efflux of chloramphenicol can be medi-

ated by the transposon Trill 696-encoded CmlA-specific

MFS transporter, identified in Pseudomonas ueruginosa (Bis- sonnette et al., 1991). Other chloramphenicol-specific MFS

transporters include the plasmid-encoded CmlB, identified in Rhodococcus fascians (Desomer et al., 1992).

High-level resistance to fluoroquinolones usually results from accumulation of several mutations affecting perme-

ability and the target enzyme DNA gyrase. It has been observed among clinical isolates of Smphylococcus aureus, Pseudomonas aeruginosa, and species of Enterobacteriaceae

(Poole, 1994; Acar and Goldstein, 1997). In Pseudomonas aeruginosa, the broad specificity multidrug transporter, the

MexA-MexB-OprM complex from the RND family, contributes to resistance not only to quinolones, but also to tet-

racycline, chloramphenicol and p-lactams (Poole, 1994; Li et al., 1994, 1995). In Smphylococcus uureus, the efflux of quinolones, chloramphenicol and puromycin is mediated by the MFS transporter NorA (Kaatz and Seo, 1995).

Interestingly, tetracycline resistance is also mediated by other broad-spectrum MDR transporters, including the hu-

man P-gp, product of the MDRl gene (Kavallaris et al.,

1993) and the yeast Saccharomyces cerevisiae Pdr5p and

Yorlp.* PdrSp also mediates resistance to chloramphenicol (Meyers et al., 1992; Leonard et al., 1994) and Yorlp to erythromycin.*

Multiple resistance to some antiseptics and disinfectants is mediated by Smr (known also as Ebr/QacC/QacD) (Schuldiner et al., 1997) from the SMR family, which is en-

coded by a variety of plasmids from clinical isolates of Sta- phylococcus aureus and other staphylococci (Littlejohn et

al., 1992; Leelapom et al., 1994, 1995). Also, the MFS transporter QacA, often found on staphylococcal plasmids, confers resistance to antiseptics and disinfectants (Little- john et al., 1992; Leelaporn et al., 1994; Behr et al., 1994).

Of many other bacterial MDR transporters now identified, all but one are secondary transporters energized by the

proton motive force (Paulsen et al., 1996a,b). The only

ATP-driven one is the LmrA of Lactococcus lactis, which shows homology to human P-gp (van Veen et al., 1996). The clinical significance of these transporters isolated from pathogenic strains, however, is not yet clear, due partly to the fact that in most cases, their reported drug-resistance

profiles are limited to a few compounds, often not including even the most important groups of clinically used antibiotics. The level of resistance conferred by these transporters is

often low. However, the presence of transport systems, even when conferring low levels of resistance to antibiotics, in-

creases the frequency of mutations to higher-level resistance (Takiff et al., 1996; Markham and Neyfakh, 1996). This can result from combination of efflux with other resistance mechanisms, or increased efficiency of efflux due to overproduction or mutation of the transporters. Duplica- tion of Smr, for example, on a transferable pTZ22 plasmid of Staphylococcus uureus doubled the efflux rate and con-

*Kolaczkowski, M. et al. In viuo screening of the substrate specificity of the yeast multidrug resmance network. Manuscript in preparatim.


ferred high-level resistance to antiseptics. This mechanism is analogous to that found in multidrug-resistant cancer cells, which often overproduce P-gp and MRP. A recent report showing MDRl P-gp-mediated resistance to complement-mediated cytotoxicity, which was reversible by vera- pamil and anti-P-gp antibody UIC2 and HYB-241 F(ab)‘* fragments, also merits attention (Weisburg et al., 1996). It suggests that bacterial MDR transporters that share overlapping substrate specificity with MDRl, might also contribute to protection of microbes against complement- mediated lysis by the host immune system. In addition, they may also contribute to protection against the innate antimicrobial peptide-mediated immunity because many hydrophobic peptides have also been identified in the resistance spectrum of mammalian P-gps (Gottesman and Pastan, 1993).

2.7.2.2. Parasitic protozoa. The resistance of parasitic protozoa to available chemotherapeutic agents (Borst and Ouelette, 1995; Ullman, 1995; Rubio and Cowman, 1996) is alarming, in particular in Plasmodium fakiparum, which is the causative agent of malaria responsible for killing between 1.5 and 2.7 million people yearly (Butler, 1997). Re- sistance to chloroquine in Plasmodium fakiparum results mainly from decreased cellular accumulation (Pussard and Verdier, 1994). Increased active efflux of chloroquine has been observed in resistant PIasmodium falciparum and has been proposed to account for the resistance phenotype (Krogstad et al., 1987, 1992), probably resulting from amplification of the n&-like genes, homologues of mammalian P-gps (Wilson et al., 1989; Foote et al., 1989) that have been identified in this parasite. Neither the amplification of the pfmdrl gene nor efflux of chloroquine, however, segre- gated with chloroquine resistance in a genetic cross (Wellems et al., 1990). The resistance locus identified by this approach, mapping to chromosome 7 (Wellems et al., 1991), has not been cloned. Expression in Chinese hamster ovary cells of the pfmdrl gene from chloroquine-sensitive parasites conferred hypersensitivity to chloroquine, associated with its enhanced accumulation in lysosomes and their increased acidification, in contrast to the mutant alleles from chloroquine-resistant isolates, which failed to confer chloroquine hypersensitivity (Van Es et al., 1994a,b). This observation, along with the failure of photoactivatable chloroquine analogues to bind to Pghl (the protein encoded by the pfmdrl gene) (Foley et al., 1994), and the fact that chloroquine is a weak base accumulating in acidified liposomes (Cullis et al., 1991) suggest that Pghl might affect its intralysosomal concentration indirectly by pH alterations. These data confirm that mutations in Pghl, previously observed to be linked to chloroquine resistance (Foote et al., 1990), indeed can contribute to chloroquine resistance. In addition, chloroquine selection of Plasmodium fakiparum lines with amplified pfmdrl leads to its deamplification and reduced production of Pghl, but unexpectedly increases sensitivity to mefloquine (Barnes et al., 1992). In- deed, amplification of the pfmdrf gene has been correlated with increased resistance to mefloquine, quinine, and halo-

fantrine (Wilson et al., 1993; Cowman et al., 1994). Inter- estingly, Pghl shares with mammalian P-gps and MRP the ability to partially complement the STE6 deficiency of Sac- charomyces cereoisiae, suggesting that it is able to transport the yeast a-mating factor (Volkman et al., 1996; Raymond et al., 1992; Ruetz et al., 1996). Thus, the mechanism of Pghl function seems to be quite complex.

Recently, the presence of a transport system mediating chloroquine uptake and defective in several chloroquine- resistant Plasmodium falciparum isolates, has been reported. The decreased rate of chloroquine uptake was linked to chloroquine resistance in a genetic cross (Sanchez et al., 1997).

An Entamoeba histolytica (pathogen responsible for dys- entery and liver abscesses) mutant resistant to emetine and showing increased efflux activity, cross resistance to some other hydrophobic drugs, reversible by P-gp antagonist ver- apamil, and over-expression of a P-gp homologue, has been selected in the laboratory (Samuleson et al., 1990; Desco- teaux et al., 1992).

In Leishmania spp. and Trypanosoma spp. resistance to pentavalent antimonials, which are drugs of choice for the treatment of espundia, kala azar (caused by L.&mania spp.), and sleeping sickness (caused by Trypanosoma spp.), is often observed in clinical isolates (Ouelette and Papa- dopoulou, 1993; Bacchi, 1993). Increase in antimonial efflux was observed in selected mutants of Leishmania spp., which often amplify parts of their genome as extrachromo- somal circles. These circles contain several repeated sequences and drug-resistance genes (Ouelette and Papa- dopoulou, 1993). One of these is the P-gp-related gene &@A, which was shown by transfection experiments to confer only low-level resistance to oxyanions in the form of pentavalent antimony compounds. The decreased accumulation of 73As 0 2 in the cells, due to @@A expression, was not observed however (Papadopoulou et al., 1994).

2.7.2.3. Fungi. The number and variety of antifungal agents is inferior to the plethora of antibacterial antibiotics. The therapeutic options for treating fungal infections, often caused by the emerging new pathogens whose incidence has increased due to the AIDS pandemic and use of immu- nosuppressive drugs in transplant and cancer patients, are limited by the relatively low number and structural variety (as compared with antibacterial antibiotics) of antifungals developed in the last decades. Additional limitations are imposed by their selectivity, pharmacokinetic profiles, and side effects (Como and Dismukes, 1994; Georgopapadakou and Walsh, 1994; Vanden Bossche, 1995; Hazen, 1995; Tu- ite, 1996). The antifungal agents effective against life- threatening infections of deep tissues comprise the three azoles fluconazole, itraconazole, and ketoconazole, and 5-fluorocytosine and amphotericin B (Como and Dis- mukes, 1994). For some infections, however, there is no effective therapy. It is worrying that the efficacy of existing antifungals is threatened by shifting of the population of pathogenic fungi towards the intrinsically resistant species and development of resistance in the species usually re-


garded as sensitive (Price et al., 1994; Vanden Bossche et

al., 1994; Marichal and Vanden Bossche, 1995; Hazen,

1995; Odds, 1996).

Although active efflux as a mechanism of fungal pathogen resistance has been appreciated only recently, it has been suggested as an important mechanism of resistance to fluconazole (DuPont et al., 1996). Multidrug transporters of the MFS-type such as CaMDRl/Benr (Fling et al., 1991)

and ABC-type such as CDRl (Prasad et al., 1995) and CDR2 (Sanglard et al., 1997) conferring a broad spectrum drug resistance (see Table l), have been identified in the principal human yeast pathogen Candida &cans. CDRl and CDR2 turned out to be homologues of the previously

identified Snq2p and Pdr5p of the yeast Saccharomyces cerevisiae (Servos et al., 1993; Balzi et al., 1994; Bissinger and Kuchler, 1994; Hirata et al., 1994) (56% identity between

CDRl and PdrSp and 42% identity between CDRI and

Snq2). Snq2p and Pdr5p are the first identified members of a distinct subgroup of ABC transporters, characterized by

their inverted domain organization in the primary sequence. These resemble the mirror image of the mammalian P-gps in that they contain a tandem repeat of an ATP-binding domain (NBD) followed by six TM helices (NBD-TM- NBD-TM). In contrast, the primary structure of mamma-

lian P-gps can be abbreviated TM-NBD-TM-NBD (Decot- tignies and Goffeau, 1997). ABC transporters of this type

have only been reported so far in fungi and plants, and also

include AtrA and AtrB of the model filamentous fungus Aspergillus nidulans (Del Sorbo et al., 1997). AtrB also confers MDR in the heterologous host Sacchuromyces cereplisiae.

Importantly, various filamentous fungi, including Aspergil- lus species, are also responsible for a variety of human infec-

tions (Vanden Bossche, 1995), including pulmonary aspergillo- sis, which is a leading cause of mortality in bone marrow transplant recipients. In Candida al&cans CaMDRl/Benr, CDRl or CDR2 deletions lead to hypersensitivity to many

drugs, including the most important fungicides in clinical use (Goldway et al., 1995; Sanglard et al., 1996, 1997). Their over-expression has been observed in resistant clini-

cal isolates (Sanglard et al., 1995, 1997; Albertson et al.,

1996). Activity of MDR transporters, at least in part, may account for the general low permeability of many fungal

pathogens, such as different Candida species, to a variety of inhibitors of potential antifungal targets. Many such inhibitors show poor intracellular accumulation and cannot be developed as antifungal drugs. This permeability barrier is reminiscent of the long-known MDR phenomenon called pleiotropic drug resistance (PDR) observed in the non-

pathogenic yeast Saccharomyces cerevisiae (Balzi and Gof- feau, 1995; Goffeau et al., 1997).

3. THE DILEMMA OF

BROAD SUBSTRATE SPECIFICITY

The common and intriguing feature of MDR transporters in contrast to their non-MDR counterparts is their surpris-

ingly broad substrate specificity. The resistance mecha-

nisms discussed in Section 2 are usually limited to sets of

closely structurally related molecules with a similar mode of

action. Even though a few specific mechanisms can accumulate together and confer an MDR phenotype, it is not comparable with the wide resistance profile of a single MDR transporter.

3.1. Drug-Resistance Profiles of

Mu&drug-Resistance Transporters

The most intensively investigated MDR transporters in this respect are the mammalian P-gps. Their substrate specific-

ity profile includes a huge variety of hydrophobic compounds, ranging from peptides and steroid hormones to an-

ticancer drugs, such as daunorubicin, doxorubicin, vinblastine, vincristine, taxol, dactinomycin, etoposide, teniposide, and

others (Gottesman and Pastan, 1993). Understanding of this profile comes ‘mainly from comparisons of drug-resis-

tance profiles of multidrug-resistant cell lines that overexpress P-gp, selected on increasing concentrations of different cytotoxic agents, to their sensitive parental lines.

Only a few reports, however, deal with P-gp transfectants not exposed to cytotoxic drug selection. It is known that

such selection can favor mutant transporters with altered

specificity profiles (Choi et al., 1989), and can also lead to over-expression of other transporters with overlapping speci-

ficity and other resistance mechanisms not necessarily associated with efflux. Different research groups use cell lines

from different tissues, and even species (human, mice, hamster), usually selected by different procedures, while each

study reports on the effects of relatively few metabolic inhibitors. This is why it is difficult to qualitatively and quan- titatively estimate the full contribution of human MDRl

P-gp to resistance to these compounds. The only large-scale screen reported so far is that carried out by the National Cancer Institute (Bethesda, MD, USA), but only a small

part of their results have been published (Lee et al., 1994; Alvarez et al., 1995). This attempt was made to predict

P-gp substrates by correlation of mdr-1 expression and cy-

closporin A (P-gp antagonist) reversible rhodamine 123 efflux with cytotoxicity data of more than 30,000 compounds on 60 human tumor cell lines from the National Cancer lnsti-

tute drug screen database. The information is then applied to identification and characterization of P-gp antagonists of potential clinical application as MDR-reversing agents.

There are surprisingly few transporters reported for which a large number of compounds was used to generate

the drug-resistance profiles directly for instance, by comparison of cells transformed with the transporter expressing vectors with ones transformed with control vectors or by comparison of transporter expressing cells with cells in which the transporter-encoding gene has been deleted. These are the MFS-type CaMDRl/Benr of Candida al&cans, MdfA of Escherichia cob, as well as the ABC-type CDRl, CDR2 of Candida a&cans and their Saccharomyces cerevisiae Pddp homologue (see Table 1).


TABLE 1. Overlapping Drug Resistance Profiles of Candidu albicans, Sacchmomyces cerevisiue, and Escherichia coli MDR Tranmorters

candida albicuns Saccharomyces cerwisiae Escherichia coli

CaMDR 11 CDRl CDR2 PdrSp2 Snq2p Yorlp’ Atrlp4 Sgelp MdfA

MFS 1 ABC MFS

3-Amino-1,2,4,-triazole Amphotericin B Amorolfine Antimycin Benomyl Brefeldin A Camptothecin CCCP Cerulenin Compactin Chloramphenicol Crystal violet Cycloheximide Daunorubicin Dinitrophenol Erythromycin Ethidium bromide Filipin Fluconazole Fluphenazine Itraconazole Ketoconazole Miconazole Nitrogen mustard 4-Nitroquinoline-N-oxide Nystatin Oligomycin l,lO-Phenantroline Rhodamine 6G Staurosporine Sulfometuron methyl Terbinafine Triaziquone

X6 x6 x7 R7 - -

R6 X’O

R’O R7 - -

R’O R7 - - - R’S x7 X7 R’s R’S

- G - - z - X’S R’O R’O R’O R’O X’O R’O X’O R’O X6 R’5

Rl8 - X’O X6 X’5

R’O x7 x7 R’

R20 ;

R7 R7 - -

- - R7 -

x7

R7 -

-

R7

R7

R7 -

R7

R7

R7

R7 -

-

x7 -

x7

R7 -

x7

R7

- -

R* X9

R” X’2 R’3

R” R’ -

R9

R’2

Rs X8

6

R’s R’O R’O -

G

x9 -

R’2 R’j R”

-

- - -

R”

X’3 X’3 -

-

X’3

- - -

R’O R’3

R’O R’O

X20 R*O

R22

R’3 R22 -

R20

- - - - Xl’

Xl4 - -

X’4

- -

Xl4 -

-

5’

RI4

- - -

R5 - -

X5

- - - -

X5

x5

x5

-

-

-

-

R2’ -

x5

-

-

-

- - - - - x4 -

- R’6

R’7 - - R’s

-

R’6 R’9 R’6 -

- - R’6 - -

-

R, confers resistance; X, not shown to confer resistance; -, not tested; CCCP, carbonyl cyanide-m-chlorophenylhydrazone. ‘CaMDRl/Benr also confers resistance to benzimidazole-2-yl-carbamate, l-benzoyl benztriazole, I-(2’sfluoryl)-5-trifluormethyl benztriazole, and metho-

trexate, but not to l-deaza-7,8-dihydropteridine, echinocandin, 5-fluorocytosine, hygromycin, indole propyl carbamate, papulocandin, or parafluorophenyl- alaline (Fling et nl., 1991).

Tddp also confers resistance to doxorubicin, deoxycorticosterone, ionophore A23187, monensin, nigericin, progesterone, rhoadmine 123, tamoxifen, tri- fluoperazine (Kolaczkowski et al., 1996), sporidesmin (Bissinger and Kuchler, 1994), 1’ mcomycin, and venturicidin (Meyers et al., 1992), but not to FCCP (Kolaczkowski et al., 1996). Pdr5p modifies intracellular accumulation of corticosterone, dexamethasone, triamcinolone acetonide (Kralli ec al., 1995), and, together with SnqZp, estradiol (Mahe et al., 1996a).

3Yorlp also confers resistance to acetic acid, benzoic acid, cadmium chloride, leptomycin B, propionic acid, reveromycin A, and tautomycin (Cui er al., 1996). 4Atrlp has not been shown to confer resistance to canavanine, ethionine, p-fluorophenylalanine, triazolealanine, or vinblastine (Kanazawa et al., 1988).

MdfA also confers resistance to benzalkonium, ciprofloxacin, kanamycin, neomycin, norfloxacin, puromycin, rifampin, tetracycline, and tetraphenylphosphonium, but not to nalidixic acid, methyl viologen, or spectinomycin (Edgar and Bibi, 1997).

5Kanazawa et al. (1988). 6Fling et nl. (1991). 7Sanglard et al. (1997). RMeyers et al. (1992). 9Leppert et al. (1990). ‘OSanglard et al. (1995). “Reid et al. (1997). l*Kolaczkowski et al. (1996). “Hirata et al. (1994). ‘4Cui et al. (1996). ‘Trasad et al. (1995). ‘6Edgar and Bibi (1997). “Ehrenhofer-Murray et nl. (1994). ‘“Ben-Yaacov et al. (1994). ‘“Amakasu et ai. (1993). 2”Haase et al. (1992). 21Gompel-Klein and Brendel (1990). **Servos et al. (1993).

Drug Resistance Mechanisms

3.2. Mechanism

A still unresolved question can be asked at this point: How can a single transporter mediate resistance to so many different compounds?

Most of the information available on this matter has been obtained with the mammalian P-gps. Additional information comes from the analysis of other transporters, since their drug-resistance profiles often overlap with that of P-gp, suggesting functional similarity. Several lines of evidence suggest that P-gp somehow can directly recognize and export from the cell membrane the variety of compounds to which it mediates resistance. Alternative to this or the classical pump model hypothesis (Fig. 2), attempts to explain the huge spectrum of resistance, assuming indirect drug redistribution in response to P-gp-dependent changes in membrane potential or intracellular pH, have also been proposed (Roepe, 1997). A recent report by Ruetz and Gros (1994a), however, shows that the mouse Mdrla P-gp expressed in yeast secretory vesicles can mediate vinblastine transport and is not affected by changes in membrane potential and pH gradient. In addition, Mdrla I’-gp also transports the lipophilic cation tetraphenylphosphonium against

229

the inside positive membrane potential of almost 90 mV and transports colchicine against its concentration gradient (Ruetz and Gros, 1994a,b). The transport of Hoechst 33342 by purified reconstituted P-gp was not accompanied by changes in the intraliposomal pH (Shapiro and Ling, 1995). Comparable rates of ATP hydrolysis and substrate (valinomycin s6Rb+ complex) transport has also been observed recently with the hamster P-gp reconstituted in pro- teoliposomes (Eytan et al., 1996a).

Another argument favoring direct drug interaction and transport is the fact that many point mutations in P-gps (Gottesman et al., 1995) and other MDR transporters (Kly- achko et al., 1997) specifically alter the drug-resistance profiles, often increasing the level of resistance mediated to certain drugs and decreasing resistance to others, which suggests the existence of some structural preferences imposed by the mutant transporters (Gottesman et al., 1995). Altered photoaffinity labeling of mutant P-gps has also been observed (Kajiji et al., 1993). The mutagenesis and photolabeling studies pointed out the importance of TM re- gions in substrate recognition (Pawagi et al., 1994; Gottes- man et al., 1995).

embrane insertion

t

flip-flop (slow)

membrane release

CYTOPLASM FIGURE 2. Possible routes of multidrug transport. According to the “vacuum cleaner” hypothesis, drugs may be extruded directly from the membrane (inner or outer leaflet) into the extracellular space or flipped from the inner to the outer leaflet (flippase model). Direct extrusion from the cytoplasm into the extracellular space cannot be excluded (classical pump model). Transport can be energized by ATP hydrolysis (ABC transporters) or proton motive force (MPS, RND, and SMR transporters).


Support for the idea that P-gp recognizes the drugs directly from the lipid phase came from energy transfer experiments with daunorubicin (a fluorescent P-gp substrate) and the photoaffinity label iodonaphthalene azide (Raviv et al., 1990). This view was further supported by the observations that P-gp prevents the intracellular access of ace- toxymethyl (AM) esters of BCECF (BCECF-AM-2’,7’-bis- (2-carboxyethyl)-5-(and-6)-carboxyfluorescein AM ester) and other dyes, which become brightly fluorescent upon reaching the cytoplasm and upon hydrolysis by intracellular esterases (Homolya et al., 1993; Ho110 et al., 1994). Similar observations have been made with multidrug-resistant mutant cells of Luctococcus lactis. These cells also extrude another lipophilic fluorescent probe I-[4-( trimethylamino) phenyl]e6-phenylhexa-1,3,5triene (TMA-DPH), and the efflux rate has been correlated with the TMA-DPH concentration in the cytoplasmic leaflet of the plasma membrane, suggesting that an MDR transporter may recognize its substrates localized in the inner leaflet (Bolhuis et al., 199613). The same observations with TMA-DPH were also made with Luctococcus lactis cells specifically over-expressing the LmrP MDR transporter, and were confirmed with plasma membrane vesicles (Bolhuis et al., 1996a). The removal of Hoechst 33342 from the membranes by purified and reconsti, tuted P-gp has been reported by Shapiro and Ling ( 1995).

These studies, however, do not fully resolve whether the drugs are extruded directly from the membrane into the extracellular medium, as proposed by the “vacuum cleaner” model (Raviv et al., 1990; Gottesman et al., 1994), or flipped from the inner to the outer membrane leaflet, from which they can diffuse outside (the flippase model) (Hig gins, 1994). They also leave a possibility that at least some drugs are recognized directly in the cytoplasm and extruded outside (classical pump model) (see Fig. 2). The flippase mechanism was also proposed earlier for proteins responsible for maintaining the phospholipid asymmetry in biological membranes (Zachowski, 1993). Indeed, the human MDRl and mouse Mdrla recently have been shown to translocate a range of short acyl chain phospholipids (van Helvoort et al., 1996), and their close homologues not involved in MDR (human MDRZ, as well as mouse Mdr2) translocate phosphatidylcholine into the bile (Oude Elf- erink et al., 1997).

It is not understood how the mammalian P-gps and the Plasmodium falcipurum Pghl transport the relatively big, as compared with drugs, a mating factor, which was proposed as a mechanism of complementation of the defect in the yeast Succharomyces cerevisiae STM a-mating factor-specific transporter (Raymond et al., 1992; Ruetz et al., 1996; Volkman et al., 1996).

Recent data on low-resolution three-dimensional structures of hamster P-gp suggest that it forms a central membrane-spanning chamber closed at the cytoplasmic side and open to the extracellular space but large enough to allow passage of known P-gp substrates. This chamber within the membrane has an opening to the lipid phase, which might be the putative substrate-binding site through which drugs

potentially may gain access to the central pore and extracellular space (Rosenberg et al., 1997). Since the substrates of P-gps and many related transporters are hydrophobic, they tend to accumulate in membranes. It seems that by direct extrusion of these compounds from the lipid phase, the transporters not only prevent drugs from accessing their intracellular targets, but in parallel, also may protect against the adverse effects of these lipophilic compounds on membrane integrity. What determines the broad specificity of transport is still an open question.

Yet another intriguing aspect of MDRl is its ability to regulate chloride channels (Higgins, 1995; Jentsch and Gunther, 1997). Interestingly, a homologous ABC transporter, the CFTR (Zielenski and Tsui, 1995), itself functions as a chloride channel and regulator of the outwardly rectifying chloride and sodium channels (Jilling and Kirk, 1997). Similarly, another ABC transporter, the sulfonylurea receptor, regulates potassium channels (Higgins, 1995; Jentsch and Gunther, 1997). The mechanism of this regulation is not known. It might involve direct protein- protein interactions or the outflow of a soluble intermediate. ATP was suggested as a possible intermediate, but its transport by the above ABC transporters is a matter of con- troversial debate (Al-Awqati, 1995; Abraham et al., 1997; Reddy et al., 1997; Grygorczyk and Hanrahan, 1997a,b). In this context, it is interesting to note that the yeast MDR transporter genes PDR.5 and SNQ.2 have been shown to be induced by ionic stress imposed by Na+, Li+, and Mn++ cations. Deletion of these genes leads to hypersensitivity and slightly increased intracellular accumulation of these cations (Miyahara et al., 199613).

3.3. Substrate Recognition by

Multidrug Resistance-Associated Protein

MRP substrate recognition and transport is also complex. In membrane vesicles, MRP transports negatively charged endogenous glutathione conjugates, such as LTC,, LTD4, LTE,, and glutathione conjugates of lipophilic compounds, such as 2,4-dinitrophenol (Leier et al., 1994; Muller et al., 1994), ethacrynic acid (Zaman et al., 1996), monochloro melphalan (anticancer) (Jedlitschky et al., 1996), and also oxidized glutathione (Leier et al., 1996). In oiao transport of LTC,, which was competitively inhibited by alkylated glutathione derivatives, was also inhibited by taxol, VP-16, vincristine, and vinblastine, but surprisingly, this inhibition was efficient only in the presence of glutathione. Vincris- tine itself was transported by MRP in membrane vesicles in the presence of glutathione (Loe et al., 199613). MRP mediated in vitro transport of azidophenacylglutathione was competitively inhibited by oxidized glutathione, DNP-SC, the LTD, receptor antagonist 3([(3(2[7-chloro-2-quinolinyl]ethenyl)phenyl} {(3-dimethylamino-3-oxopropyl) thio) methyl] thio) propanoic acid (MK571), arsenate, daunorubicin, vincristine, and etoposide (Shen et al., 1996).

In addition to glutathione conjugates, in membrane vesicles, MRP transports glucuronides, such as 17+estradiol-


17-P-D-glucuronide (Loe et al., 1996a), glucuronosylhyode-

oxycholate, and glucuronosyletoposide, and sulfates such as sulfatolithocholyltaurine (Jedlitschky et al., 1996). Trans-

port of 17-P-estradiol-17-P-D-glucuronide was competitively inhibited by other cholestatic-conjugated steroids (Loe et al., 1996a). The transport of calcein, calcein acetomethoxym- ethyl ester, and pyrenemaleimide glutathione conjugate by MRP in whole cells has also been suggested (Feller et al., 1995; Ho110 et al., 1996). In addition to the negatively charged compounds, in membrane vesicles, MRP was shown to transport neutral or mildly cationic cytotoxic li-

pophilic drugs such as daunorubicin, vincristine, and etoposide. Daunorubicin transport was competitively inhibited

by reduced and oxidized glutathione, azidophenacylglutathione, dinitrophenyl glutathione, arsenate, genistein

and MK571 (Paul et al., 1996a,b,c). In another study, re-

duced glutathione did not inhibit LTC, transport (Loe et al., 1996b), and no MRP-mediated transport of doxorubi- tin, daunorubicin and vinblastine was observed (Jedlit- schky et al., 1996). Direct interaction of MRP with vincris-

tine, VP16, reduced and oxidized glutathione, however, was suggested by their stimulation of vanadate-induced trapping of MgATP (Taguchi et al., 1997). Finally, expression of MRP in the yeast Saccharomyces cerevisiae ste6 null

mutant partially restored mating, suggesting that MRP can

transport the a mating pheromone (Ruetz et al., 1996).

4. THE YEAST SACCHAROMYCES CEREVlSlAE PLEIOTROPIC DRUG-RESISTANCE NETWORK AS A TOOL TO UNDERSTAND THE MECHANISM OF MULTIDRUG RESISTANCE AND TRANSPORT

In the yeast Saccharomyces cerevisiae, two networks of genes involved in MDR have been identified. The first one is reg

ulated by Yaplp, a bZIP-containing transcription factor (Moye-Rowley et al., 1989) protecting yeast against oxida-

tive stress (Schnell et al., 1992; Kuge and Jones, 1994; Kuge

et al., 1997). Together with Cadlp, a Yaplp homologue, this network also protects yeast against toxic effects of cadmium, zinc, l,lO-phenanthroline, and cycloheximide (Wu et al., 1993). Yaplp also confers resistance to sulfomethu- ron methyl (Leppert et al., 1990), 4-nitroquinoline-N- oxide (4NQO), N-methyl-N’-nitro-N-nitrosoguanidine, trenimon, and triaziquone (Hertle et al., 1991; Haase et ai.,

1992). Overproduction of YapZp, the homologue of Yaplp that also specifically binds the AP-1 recognition DNA se-

quence, mediates resistance to l,lO-phenanthroline. In addition, yap2 null mutants show increased thermotolerance under iron and zinc starvation conditions induced by the chelator l,lO-phenanthroline (Bossier et al., 1993).

Cadmium resistance mediated by Yaplp requires the presence of an ABC transporter Ycflp (a homologue of MRP) (Wemmie et al., 1994), which sequesters bis(glutathionato)-cadmium in the vacuoles (Li et al., 1997). Ycfl

has been implicated additionally in the red pigment forma-

tion of Succharomyces cerevisiae a&l and ade2 mutants, pre-

sumably by transporting the glutathione conjugates of the endogenous metabolites in the adenine biosynthetic pathway (phosphoribosylaminoimidazole and phosphoribosylaminoimidazole carboxylate) into the vacuoles (Chaudhuri etal., 1996). Yaplp and Yap2p also influence the expression of PDRS and SNQ2 genes encoding ABC transporters, as their heat shock-induced expression becomes very low in

the yap], yap2 double disruptant (Miyahara et al., 1996a). The second network, called the PDR network (Balzi and

Goffeau, 1995), is regulated by the Pdrlp and Pdr3p tran-

scription factors and the unidentified PDR4, PDR7, and PDR9 loci (Dexter et al., 1994). Many spontaneously iso-

lated mutations in Pdrlp and Pdr3p result in over-expres-

sion of the ABC transporter genes SNQ2, PDRS, and YORI , which initially were cloned as genes conferring resistance to cycloheximide (Leppert et al., 1990; Balzi et al.,

1994), 4NQ0 (Haase et al., 1992; Servos et al., 1993), and oligomycin (Katzmann et al., 1995; Cui et al., 1996), re- spectively. On the other hand, the double deletion of both PDRI and PDR3 genes results in drug hypersensitivity and

strongly reduced expression of SNQ2, PDRS, and YORl (Decottignies et al., 1995; Mahe et al., 1996b; Katzmann et al., 1995).

Two other MDR transporter-encoding genes belonging

to the MFS, energized by proton motive force, have been identified in yeast. These are ATRl, conferring aminotriazole and 4NQO resistance (Kanazawa et al., 1988; Go-

mpel-Klein and Brendel, 1990), and SGE1, conferring resistance to crystal violet and ethidium bromide (Amakasu et al., 1993; Ehrenhofer-Murray et al., 1994). The transcript

of /&i”R1 has not been affected in multidrug-resistant mutants of PDR I, suggesting that it probably is not under the

control of PDRl and PDR3 (Balzi et al., 1994). Finally, sequencing of the yeast genome unraveled 13

other ABC homologues of PDRS , SNQ2, and YORl (De-

cottignies and Goffeau, 1997). This inventory revealed that

Pdr5p and Snq2p belong to a new family of ABC transporters, which so far contained no human homologues. In con-

trast, the family comprising Yorlp has several human homologues, including MRP, which shows 33% amino acid

identity to Yorlp (Katzmann et al., 1995). Also, 26 other major facilitators homologous to ATRI and SGEf have been identified in the yeast genome (Goffeau et al., 1997). Some of these are likely to be involved in multidrug transport.

The resistance profiles of the identified Saccharomyces

cerevisiae MDR transporters reported to date are summa- rized in Table 1. We have observed, however, that the

drug-resistance profile of Pddp is much larger than previously believed. It comprises many other steroids, fungicides of different chemical classes, herbicides, detergents and other toxic compounds. Its substrate specificity largely overlaps with that of Snq2p and Yorlp.” These data, ob-

*Kolaczkowski, M. et al. In uivo screennxg of the substrate specificity of the yeasr multidrug resistance network. Manuscript in preparatmn.


tained with isogenic strains in which the PDRS, ShJQ2, and YORl genes have been deleted in different combinations, allowed not only for qualitative determination of their drug-resistance profiles, but also gave gross quantitative esti- mation on the contribution of particular transporters to the

resistance toward a few hundred compounds. These observa-

tions and that of others (Mahe et al., 1996a; Sanglard et al., 1997) establish that the presence of several transporters with overlapping specificity often prevents the observation of phenotypes associated with their single deletions. Cautious

interpretation of results from drug-resistance assays, aiming at determination of drug-resistance profiles of particular

transporters, is thus required, especially for assays performed with cell lines obtained by stepwise drug selection, which

may involve several overlapping resistance determinants. In nature, the presence of such a flexible system assures very efficient protection of cells against a variety of toxic insults.

To better understand Pdr5p-mediated transport of cyto-

toxic compounds, we have developed new sensitive fluorescence-based assays, allowing for the in viva and in vitro characterization of drug transport. In particular, the sensitive in

vitro assay for ATP-dependent Pdr5p-mediated rhodamine

6G fluorescence quenching in plasma membrane prepara- tions is the first reported assay allowing for a detailed large- scale kinetic characterization of multidrug transporter in-

hibitors (Kolaczkowski et al., 1996). This assay, combined with in viva toxicity screening, provides a wealth of information for rational molecular modeling of new MDR trans-

porter antagonists. The highly overlapping specificity profiles of the yeast

MDR transporters with other prokaryotic and eukaryotic

ones (Table 1) imply some functional and mechanistic similarity. It is likely, therefore, that our understanding oI the

PdrSp mechanism of multidrug transport will contribute to

an understanding of clinically relevant transporters such as, for example, MDRl P-gp or the PdrSp homologues CDRl

and CDR2 involved in MDR of the pathogenic yeast Cun- dida albicans. It is likely that our transport assay could be also functional with heterologous MDR genes expressed in

yeast plasma membranes. Heterologous expression of ABC proteins has been achieved in Saccharomyces cerevisiae (Kuchler and Thorner, 1992; Ruetz and Gros, 1994a), but the level of over-expression is often limiting. The huge over-expression of PDR5 in the regulatory mutants of PDRl

and PDR3, and the availability of strains deleted in the en-

dogenous yeast transporters, makes it an attractive system for overproduction, under the control of the PDR5 pro- moter, of other clinically relevant ABC transporters, some involved in MDR, such as P-gp, but also others such as

CFTR and sulfonylurea receptor.

5. OTHER PRACTICAL IMPLICATIONS OF THE YEAST PLEIOTROPIC DRUG-RESISTANCE NETWORK

Information on the PDR network modifying transport of a variety of compounds can be exploited for several practical applications.

Yeast has been used for analysis of the mechanism of action of certain anticancer drugs (Nitiss and Wang, 1988; Abe et al., 1994; Fox et al., 1994; Nitiss, 1994; Kauh and

Bjornsti, 1995; Ishida et al., 1995I/Karavokyros and Deli- theos, 1997) and other drugs (Wdoden et al., 1997). In this

context, we have shown that PdrSp confers resistance to several anticancer agents (Kolaczkowski et al., 1996), ap- parently by reducing their accumulation in the cells. Re-

cently, resistance to the anticancer, topoisomerase-targeted drug camptothecin has been associated with the PDR net-

work. In particular, SnqZp, but also PdrSp, when overpro- duced, conferred some resistance to this compound (Reid et

al., 1997). Yeast has also been used for functional and mu-

tational analysis of cloned steroid hormone receptors (Lind et al., 1996). The influence of the PDR network on intracellular hormone availability interfering with the transcriptional activity of steroid hormone receptors has been indi- cated by Gilbert et al. (1993). As mentioned in Section 4, the transporters Pdr5p and Snq2p are involved in the efflux

of a large series of steroids (Kralli ec al., 1995; Mahe et al.,

1996a; Kolaczkowski et al., 1996; Kolaczkowski et al.*). Use

of more permeable yeast strains deleted in MDR transport-

ers or their regulators would eliminate their interference with these applications.

The mammalian hepatobiliary metabolism and excretion

of endo- and xenobiotics responsible for the clearance of many chemotherapeutic drugs are used to determine the pharmacokinetic behavior of compounds important in drug

development (Kling, 1996). In this respect, yeast Saccharo- myces cerewisiae serves as an alternative to mammalian cell cultures to study the metabolism of a variety of drugs and

xenobiotics by the heterologously expressed human enzymes involved in detoxification, including cytochrome

P450 (Pompon et al., 1995). Again, the active permeability barrier of the PDR network interferes with these investiga-

tions. One of the important transport systems of the hepatic

canalicular membrane, the understanding of which would be highly facilitated by cloning its gene, is the bile acid transporter. Our data* suggest that the PDR network is involved in resistance to certain bile acids, likely to be mediated by the Bat1 ABC transporter, mediating taurocholate

uptake into the secretory vesicles (St-Pierre et al., 1994; Oritz et al., 1997). Therefore, the yeast system should offer an easy screen for expression cloning of the human canalic-

ular bile acid transporter.

Development, testing, and release of new antibiotics is a costly and time-consuming process. An alternative strategy, of which the P-lactamase inhibitors (clavulanic acid, sul- bactam, tazobactam) co-administered with p-lactam antibiotics, are the first successful examples, is to overcome resistance by its inhibition (Coleman et al., 1994). Inhibition of bacterial efflux pumps has also been pursued to revitalize old antibiotics, such as tetracycline, that lost their efficacy due to widespread resistance (Service, 1995). Inhibition of

*Kolaczkowski, M. et nl. In viva screening of the substrate specificity of the

yeast multidrug resistance network. Manuscript in preparation.


P-gp is one of the strategies to increase the efficiency of chemotherapy of certain tumors, particularly of the he-

matopoietic system. Although some improvement has been observed in several cases by co-administration of P-gp antagonists with anticancer drugs, it usually suffers from many side effects. These side effects result from the toxicity of the

modulators, as well as changes in the pharmacokinetic profile of anticancer drugs, which gain easier access to the key

organs normally protected by P-gp, such as brain. Several new P-gp modulators are at the stage of clinical trial (He-

gewisch-Becker, 1996). The biochemical characterization of multidrug transport,

necessary for rational design of inhibitors, has been hin- dered by lack of convenient in vitro assays of drug transport. The assays based on uptake of radioactive substrates require high amounts of precious biological samples, suffer from

high noise, and are expensive, laborious and time-consuming; hence, they are not suitable for large-scale analysis. The only three fluorescence-based in vitro assays reported to

date, allowing monitoring of transport in real time, lack suf-

ficient sensitivity for the kinetic characterization of inhibitors (Guiral et al., 1994; Shapiro and Ling, 1995; Bolhuis et al., 1996a). Development of in vitro screening assays, such as the one described for the yeast Pdr5p (Kolaczkowski et al., 1996), allowing rapid characterization of MDR transporter inhibitors, combined with the crystallization and

generation of high-resolution three-dimensional structures, may contribute significantly to the rational design of new

MDR modulators. The hypersensitivity of yeast deleted in the regulators of

the PDR network to many chemotherapeutic drugs points

to the possibility of overcoming MDR by inhibition of regulators of MDR pumps. This is an interesting alternative to

inhibition of transporters. Yeast and other MDR transporters can also be used as in

uiuo selectable markers in expression vectors. This has implications not only for industrial fermentations, but also for gene therapy, which is hampered by inefficient DNA trans-

fer and unstable expression of transgenes (Blau et al., 1997). Among other chemoresistance genes, MDR transporters of-

fer the possibility of positive selection of transfected cells,

not only in cell culture, but also in piivo by means of drugs that have been well characterized pharmacokinetically.

This approach may improve the efficiency of gene therapy, particularly of hematopoietic disorders (Kane, 1996; Bank, 1996; Licht et al., 1997; Gottesman and Pastan, 1997; Moritz and Williams, 1997). For example, the efficiency of

expression of the nonselectable glucocerebrosidase gene has been increased by its translational fusion with MDRI, which resulted in elevation of glucocerebrosidase expression upon drug selection (Aran et al., 1996). Anticancer chemotherapy suffers from severe, dose-limiting side effects, often resulting from the toxicity to cells of the hematopoietic system. Transfusions and administration of hematopoie-

tic growth factors are applied in clinics to reduce morbidity after chemotherapy. One of the strategies to circumvent this problem is the protection of normal hematopoietic

cells by introduction of vectors expressing drug-resistance

genes such as MDRJ (Gottesman and Pastan, 1997; Moritz

and Williams, 1997). The possibility of biotransformation of compounds by

combined expression of different biosynthetic genes (Roessner and Scott, 1996) has received a recent impetus

by the development of directed DNA shuffling. It is based on generation of a large variety of different combinations of

mutations by several rounds of DNA fragmentation and primerless polymerase chain reaction, combined with spe-

cific selection for the desired traits. This technology has been applied successfully for the improvement of single gene products, but also whole operons (Crameri et al., 1997;

Wackett, 1997). It generates a large diversity of different combinations of desirable mutations, allowing, for example, the isolation of novel antibiotics not previously encoun-

tered in nature by manipulation of different genes involved in their biosynthesis. The diversity of generated new combinations of enzymes and resulting metabolites is limited,

however, by their toxicity to the producing host. One of

the strategies to overcome this problem, widely used in nature by antibiotic-producing organisms, would be to employ

MDR transporters to actively extrude such toxic metabolites outside the cells.

Recently, the production by yeast and other microorganisms of diverse surface active molecules has received con- siderable attention as biodegradable alternatives to tradi-

tional chemically synthesized surfactants. Many compounds of medicinal importance, steroids in particular, are pro- duced by microbial biotransformations, including yeast

(Ward and Young, 1990; Voishvillo et al., 1994; Mahato and Garai, 1997). Our observations, which indicate the in-

volvement of the yeast MDR transporters in resistance to several detergents,* as well as steroids, might be exploited to better control their production.

Better knowledge of the specificity of MDR transporters and the ability to manipulate them in a predictable way would be an obvious advantage in the design of particular biotransformations. Expression of transporters not affecting substrates, but extruding end products of biotransformations or unwanted intermediates to culture media, would

increase product yield and facilitate purification. There is

no reason to believe that this technology cannot be devel-

oped in yeast, using Pdr5p or other yeast MDR pumps. The yeast Saccharomyces cerevisiae can be used for presen-

tation of peptide or antibody libraries, production of recombinant vaccines and of immobilized whole cell biocatalysts.

These applications require the surface presentation of proteins or smaller peptide epitopes. Presentation of different small peptide epitopes in bacteria has been successfully achieved by fusion with the surface-exposed loops of outer- membrane proteins (Stahl and Uhlen, 1997; Georgiou et al., 1997). Due to the high overproduction and membrane localization of Pdr5p in the PDRl mutants, this protein

*Kolaczkowski, M. et al. In viva screening of the substrate specificity of the yeast multidrug resistance network. Manuscript in preparation.


might offer attractive attachment points for such applications.

In brief, due to homology with mammalian, parasite, and

other microbial resistance systems, the yeast Succhomyces cereoisiae PDR network proves to be a good model to inves-

tigate MDR mechanisms. The understanding of the mechanism of broad specificity of MDR transporters may contrib-

ute significantly to the improvement of not only clinical, therapeutic applications, but also of biotechnological biotransformations of pharmaceutical interest. These can profit from the remarkable genetic properties of Saccharomyces cerewi- sti and its “generally regarded as safe” status, which allows its use for food and pharmaceutical production.

Acknowkdgements-We wish to thank Anna Kolaczkowska, Susan Cronin, Elisabecta Balzi, and Bart van den Hazel for general support and helpful comments and discussion. This work was supported in part by grants from the Service de la Politique Scientifique: Action Sciences de la Vie and by Fonds National de la Recherche Scientifique, Belgium.

References

Abe, H., Wada, M., Kohno, K. and Kuwano, M. (1994) Altered drug sensitivities to anticancer agents in radiation-sensitive

DNA repair deficient yeast mutants. Anticancer Res. 14: 1807-

1810.

Abraham, E. H., Okunieff, P., Scala, S., Vos, P., Oosterveld, M. J.,

Chen, A. Y., Shrivastav, B. and Guidotti, G. (1997) Cystic

fibrosis transmembrane conductance regulator and adenosine

triphosphate. Science 275: 1324-1325.

Acar, J. F. and Goldstein, F. W. (1997) Trends in bacterial resis-

tance to fluoroquinolones. Clin. Infect. Dis. 24 (Suppl. 1): S67-

s73. Al-Awqati, Q. (1995) Regulation of ion channels by ABC trans-

porters that secrete ATP. Science 269: 805-806. Albertson, G. D., Niimi, M., Cannon, R. D. and Jenkinson, H. F.

(1996) Multiple efflux mechanisms are involved in Candida

albicans fluconazole resistance. Antimicrob. Agents Chemother.

40: 2835-2841.

Alvarez, M., Paull, K., Monks, A., Hose, C., Lee, J. S., Weinstein,

J., Grever, M., Bates, S. and Fojo, T. (1995) Generation of a

drug resistance profile by quantitation of mdrJ/P-glycoproetin

in the cell lines of the National Cancer Institute anticancer

drug screen. J. Clin. Invest. 95: 2205-2214.

Amakasu, H., Suzuki, Y., Nishizawa, M. and Fykasawa, T. (1993)

Isolation and characterisation of SGEJ: a yeast gene that par-

tially suppresses the gall J mutaion in multiple copies. Genetics

134: 675-683.

Anderson, P. (1997) Kinase cascades regulating entry into apopto-

sis. Microb. Mol. Biol. Rev. 61: 33-46. Aran, J. M., Licht, T., Gottesman, M. M. and Pastan, I. (1996)

Complete restoration of glucocerebrosidase deficiency in gau- cher fibroblasts using a bicistronic MDR retrovirus and a new

selection strategy. Hum. Gene Ther. 7: 2165-2175.

Arceci, R. J. (1993) Clinical significance of P-glycoprotein in mul-

tidrug resistance malignancies. Blood 81: 2215-2222.

Arthur, M., Brisson-Noel, A. and Courvalin, P. (1987) Origin and evolution of genes specifying resistance to macrolide, lincosamide and streptogramin antibiotics; data and hypothesis. J.

Antimicrob. Chemother. 20: 783-802. Awasthi, S., Singhal, S. S., Srivastava, S. K., Zimniak, P., Bajpai,

K. K., Saxena, M., Sharma, R., Ziller, S. A., III, Frenkel, E. P.,

Singh, S. V., He, N. G. and Awasthi, Y. C. (1994) Adenosine

triphosphate-dependent transport of doxorubicin, daunomy-

tin, and vinblastine in human tissues by a mechanism distinct

from the P-glycoprotein. J. Clin. Invest. 93: 958-965.

Awasthi, Y. C., Saxena, M., Sharma, R., Sharma, R., Singhal, S. S.

and Ahmad, H. (1992) Role of glutathione S-transferase and

dinitrophenyl S-glutathione ATPase (Dnp-SG ATPase) in the

protection of membranes from xenobiotics and oxidative stress.

Int. J. Toxicol. Occup. Environ. Health 1: 77-83.

Bacchi, C. J. (1993) Resistance to clinical drugs in African trypa- nosomes. Parasitol. Today 9: 190-193.

Balzi, E. and Goffeau, A. (1995) Yeast multidrug resistance: the

PDR network. J. Bioenerg. Biomembr. 27: 71-76.

Balzi, E., Wang, M., Leterme, S., van Dyck, L. and Goffeau, A.

(1994) PDR5, a novel yeast multidrug resistance conferring

transporter controlled by the transcriptional regulator PDRl. J.

Biol. Chem. 269: 2206-2214.

Bank, A. (1996) Human somatic gene therapy. Bioessays 18: 999-

1007.

Barnes, D. A., Foote, S. J., Galatis, D., Kemp, D. J. and Cowman, A. F. (1992) Selection for high-level chloroquine resistance

results in deamplification of the j$mdrJ gene and increased sensitivity to mefloquine in Plasmodium faki@rum. EMBO J. 11:

3067-3075. Behr, II., Reverdy, M. E., Mabilat, C., Frency, J. and Fleurette, J.

(1994) Relationship between the level of minimal inhibitory

concentrations of five antiseptics and the presence of qacA gene

in St@hylococcus aureus. Pathol. Biol. (Paris) 42: 438444.

Ben-Yaacov, R., Knoller, S., Caldwell, G. A., Becker, J. M. and

Koltin, Y. (1994) Candida &cans gene encoding resistance to

benomyl and methotrexate is a multidrug resistance gene. Anti-

microb. Agents Chemother. 38: 648-652.

Bissinger, P. H. and Kuchler, K. (1994) Molecular cloning of the

Succharomyces cereoisiae STSJ gene product. J. Biol. Chem. 269:

4180-4186. Bissonnette, L., Champetier, S., Buisson, J. I’. and Roy, P. H.

(1991) Characterization of the nonenzymatic chloramphenicol

resistance (&A) gene of the In4 integron of TN J 696: similar-

ity of the product to transmembrane transport proteins. J. Bac-

teriol. 173: 449311502.

Blanchard, J. S. (1996) Molecular mechanisms of drug resistance

in Mycobacterium tuberculosis. Annu. Rev. Biochem. 65: 215-

239.

Blau, C. A., Neff, T. and Papayannopoulou, T. (1997) Cytokine

prestimulation as a gene therapy strategy: implications for using

the MDRl gene as a dominant selectable marker. Blood 89:

146-154.

Bock, K. W. (1991) Roles of UDP-glucuronyl transferases in

chemical carcinogenesis. Crit. Rev. Biochem. Mol. Biol. 26:

129-150.

Bolhuis, H., van Veen, H. W., Brands, J. R., Putman, M., Pool- man, B., Driessen, A. J. and Konings, W. N. (1996a) Energetics

and mechanism of drug transport mediated by the lactococcal

multidrug transporter LmrP. J. Biol. Chem. 271: 24123-

24128. Bolhuis, H., van Veen, H. W., Molenaar, D., Poolman, B., Dries-

sen, A. J. and Konings, W. N. (1996b) Multidrug resistance in Lactococcus hais: evidence for ATP-dependent drug extrusion from the inner leaflet of the cytoplasmic membrane. EMBO J. 15: 4239-4245.

Borst, P. and Ouelette, M. (1995) New mechanisms of drug resis-

tance in parasitic protozoa. Annu. Rev. Microbial. 49: 427460.


Borst, P. and Schinkel, A. H. (1997) Genetic dissection of the

function of mammalian P-glycoproteins. Trends Genet. 13:

217-222.

Bossier, P., Fernandes, L., Rocha, D. and Rodrigues-Pousada, C.

(1993) Overexpression of YAP2, coding for a new YAP protein, and YAP1 in Succ~rom~ces cerevisisiae alleviates growth inhibition

caused by l,lO-phenantroline. J. Biol. Chem. 268: 23640-23645.

Brennan, P. J. and Nikaido, H. (1995) The envelope of mycobacteria. Annu. Rev. Biochem. 64: 2963.

Bromme, D., Rossi, A. B., Smeekens, S. P., Anderson, D. C. and

Payan, D. G. (1996) Human bleomycin hydrolase: molecular

cloning, sequencing, functional expression, and enzymatic

characterization. Biochemistry 35: 6706-6714.

Brown, A. J. and Richman, D. D. (1997) HIV-l: gambling on the

evolution of drug resistance. Nat. Med. 3: 268-271.

Butler, D. (1997) Time to put malaria on the global agenda.

Nature 386: 535-536.

Chaudhuri, B., Ingavale, S. and Bachhawat, A. K. (1996) &pl +, a

gene required for red pigment formation in c&6 mutants of

Schixosaccharomyces pombe, encodes an enzyme required for glutathione biosynthesis: a role of glutathione and a glutathione-

conjugate pump. Genetics 145: 75-83.

Chen, G., Duran, G. E., Steger, K. A., Lacayo, N. J., Jaffrezou,

J. P., Dumontet, C. and Sikic, B. I. (1997) Multidrug-resistant

human sarcoma cells with a mutant P-glycoprotein, altered pheno-

type, and resistance to cyclosporins. J. Biol. Chem. 272: 5974-5982.

Choi, K., Chen, C. J., Kriegler, M. and Roninson, I. B. (1989) An

altered pattern of cross-resistance in multidrug-resistant human

cells results from spontaneous mutations in the m&l (P-glyco-

protein) gene. Cell 53: 519-529.

Cohen, B. E. (1992) A sequential mechanism for the formation of

aqueous channels by amphotericin B in liposomes: the effect of sterols and phospholipid composition. Biochem. Biophys. Acta

1108: 49-58.

Cole, S. P., Bhardwaj, G., Gerlach, J. H., Mackie, J. E., Grant,

C. E., Almquist, K. C., Stewart, A. J., Kurz, E. U., Duncan,

A. M. V. and Deeley, R. G. (1992) Overexpression of a trans-

porter gene in a multidrug resistant human lung cancer cell

line. Science 258: 1650-1654.

Coleman, K., Athalye, M., Clancey, A., Davison, M., Payne, D. J.,

Perry, C. R. and Chopra, I. (1994) Bacterial resistance mecha-

nisms as therapeutic targets. J. Antimicrob. Chemother. 33:

1091-1116.

Commandeur, J. N., Stijntjes, G. J. and Vermeulen, N. P. (1995)

Enzymes and transport systems involved in the formation and

disposition of glutathione S-conjugates. Pharmacol. Rev. 47:

271-330.

Como, J. A. and Dismukes, W. E. (1994) Oral azole drugs as sys-

temic antifungal therapy. N. Engl. J. Med. 330: 263-272.

Cowman, A. F., Galatis, D. and Thompson, J. K. (1994) Selection

for mefloquine resistance in Plasmodium falciparum is linked to

amplification of the pfmdrl gene and cross-resistance to halo-

fantrine and quinine. Proc. Natl. Acad. Sci. USA 91: 1143-

1147. Crameri, A., Dawes, G., Rodriguez, E., Jr., Silver, S. and Stemmer,

W. P. (1997) Molecular evolution of an arsenate detoxification

pathway by DNA shuffling. Nat. Biotechnol. 15: 436-438. Cui, Z., Hirata, D. Tsuchiya, E., Osada, H. and Miyakawa, T.

(1996) The multidrug resistance-associated protein (MRP) subfamily (Yrsl/Yorl) of Saccharomyces cereoisiae is important

for the tolerance to a broad range of organic anions. J. Biol. Chem. 271: 14712-14716.

Cullis, P. R., Bally, M. B., Madden, T. D., Mayer, L. D. and Hope,

M. J. (1991) pH gradients and membrane transport in liposomal

systems. Trends Biotechnol. 9: 268-272.

Davies, J. (1994) Inactivation of antibiotics and the dissemination

of resistance genes. Science 264: 375-382.

Decottingies, A. and Goffeau, A. (1997) Complete inventory of the yeast ABC proteins. Nat. Genet. 15: 137-145.

Decottignies, A., Lambert, L., Catty, I’., Degand, H., Epping,

E. A., Moye-Rowley, W. S., Balzi, E. and Goffeau, A. (1995)

Identification and characterization of SNQZ, a new multidrug

ATP binding cassette transporter of the yeast plasma mem-

brane. J. Biol. Chem. 270: 18150-18157.

Del Sorbo, G., Andrade, A. C., Van Nisterlooy, J. G., Van Kan,

J. A., Balzi, E. and De Waard, M. A. (1997) Multidrug resis-

tance in AspergiUus nid&ns involves novel ATP-binding cas-

sette transporters. Mol. Gen. Genet. 254: 417-426.

Descoteaux, S., Ayala, P., Orozco, E. and Samuelson, J. (1992)

Primary sequences of two P-glycoprotein genes of Entamoebu

histolyticu. Mol. B&hem. Parasitol. 54: 201-212.

Desomer, J., Vereecke, D., Crespi, M. and Van, M. M. (1992) The

plasmid-encoded chloramphenicol-resistance protein of Rhodo-

coccus fascians is homologous to the transmembrane tetracy-

cline efflux proteins. Mol. Microbial. 6: 2377-2385.

Dexter, D., Moye-Rowley, W. S., Wu, A. L. and Golin, J. (1994)

Mutations in the yeast PDR3, PDR4, PDR7 and PDR9 pleiotro-

pit (multiple) drug resistance loci affect the transcript level of

an ATP binding cassette transporter encoding gene, PDRS.

Genetics 136: 505-515.

Doige, C. A. and Ames, G. F. (1993) ATP-dependent transport

systems in bacteria and humans: relevance to cystic fibrosis and

multidrug resistance. Annu. Rev. Microbial. 47: 291-319.

DuPont, B. F., Dromer, F. and Improvisi, L. (1996) The problem of

azole resistance in Candida. J. Mycol. Med. 6 (Suppl.): 12-18.

Edgar, R. and Bibi, E. (1997) MdfA, an Escherichia coli multidrug

resistance protein with an extraordinarily broad spectrum of

drug recognition. J. Bacterial. 179: 2274-2280.

Ehrenhofer-Murray, A. E., Wurgler, F. E. and Sengstag, C. (1994)

The Sacchromyces cereuisiae SGEJ gene product: a novel drug-

resistance protein within the major facilitator superfamily. Mol.

Gen. Genet. 244: 287-294.

Eytan, G. D., Regev, R. and Assaraf, Y. G. (1996a) Functional

reconstitution of P-glycoprotein reveals an apparent near sto-

ichiometric drug transport to ATP hydrolysis. J. Biol. Chem.

271: 3172-3178.

Eytan, G. D., Regev, R., Oren, G. and Assaraf, Y. G. (1996b) The

role of passive transbilayer drug movement in multidrug resis-

tance and its modulation. J. Biol. Chem. 27 1: 12897-l 2902.

Falany, C. N. (1991) Molecular enzymology of human liver cyto-

solic sulfotransferases. Trends Pharmacol. Sci. 12: 255-259.

Fath, M. J. and Kolter, R. (1993) ABC transporters: bacterial

exporters. Microbial. Rev. 57: 995-1017.

Feller, N., Broxterman, H. J., Wahrer, D. C. and Pinedo, H. M.

(1995) ATP dependent efflux of calcein by the multidrug resis-

tance protein (MRP): no inhibition by intracellular glutathione

depletion. FEBS Lett. 368: 385-388. Filipits, M., Suchomel, R. W., Zochbauer, S., Malayeri, R. and

Pirker, R. (1996) Clinical relevance of drug resistance genes in malignant diseases. Leukemia 10 (Suppl.): SlO-S17.

Fling, M. E., Kopf, J., Tamarkin, A., Gorman, J. A., Smith, H. A.

and Koltin, Y. (1991) Analysis of Candida albicans gene that encodes a novel mechanism for resistance to benomyl and

methotrexate. Mol. Gen. Genet. 227: 318-329.


Foley, M., Deady, L. W., Ng, K., Cowman, A. F. and Tilley, L. (1994) Photoaffinity labelling of chloroquine-binding proteins in Plasmodiumfafcipar~m. J. Biol. Chem. 269: 6955-6961.

Foote, S. J., Thompson, J. K., Cowman, A. F. and Kemp, D. J, (1989) Amplification of the multidrug resistance gene in some chloroquine-resistant isolates of Plasmodium falciparum. Cell 57:

921-930. Foote, S. J., Kyle, D. E., Martin, R. K., Oduola, A. M., Forsyth, K.,

Kemp, D. J. and Cowman, A. F. (1990) Several alleles of the multidrug resistance gene are closely linked to chloroquine resistance in Plasmodium fulciparum. Nature 345: 255-258.

Fox, M. E., Feldman, B. J. and Chu, G. (1994) A novel role for DNA photolyase: binding to DNA damaged by drugs is associated with enhanced cytotoxicity in Saccharomyces cereuisiae. Mol. Cell. Biol. 14: 8071-8077.

Geerts, S., Coles, G. C. and Gryseels, B. (1997) Anthelmintic resistance in human helminths: learning from the problems with worm control in livestock. Parasitol. Today 13: 149-151.

Georgiou, G., Stathopoulos, C., Daugherty, P. S., Nayak, A. R., Iverson, B. L. and Curtiss, R., III (1997) Display of heterologous proteins on the surface of microorganisms: from the screening of

combinatorial libraries to live recombinant vaccines. Nat. Bio- technol. 15: 2940.

Georgopapadakou, N. K. and Walsh, T. J. (1994) Human mycoses: drugs and targets for emerging pathogens. Science 264: 371-

373. Gilbert, D. M., Heery, D. M., Losson, R., Chambon, P. and Le-

moine, Y. (1993) Estradiol-inducible squelching and cell growth arrest by a chimeric VPl6-estrogen receptor expressed in Snccharomyces cereuisiae: suppression by an allele of PDRI. Mol. Cell. Biol 13: 462472.

Goffeau, A., Park, J., Paulsen, 1. T., Jonniaux, J. L., Dinh, T., Mor- dant, P. and Saier, M. H., Jr. (1997) Multidrug-resistant trans-

port proteins in yeast: complete inventory and phylogenetic characterization of yeast open reading frames within the major facilitator superfamily. Yeast 13: 43-54.

Gold, H. S. and Moellering, R. C. (1996) Antimicrobial-drug

resistance. N. Engl. J. Med. 355: 1445-1453. Goldman, R. C. and Cappobianco, J. 0. (1990) Role of an energy-

dependent efflux pump in plasmid pNE24-mediated resistance to 14- and 15-membered macrolides in Staphylococcw epdermi- dis. Antimicrob. Agents Chemother. 34: 1973-1980.

Goldway, M., Teff, D., Schmidt, R., Oppenheim, A. B. and Kol- tin, Y. (1995) Multidrug resistance in Candidu &cans: disruption of the Benr gene. Antimicrob. Agents Chemother. 39: 422426.

Compel-Klein, P. and Brendel, M. (1990) Allelism of ShJQI and

ATRl genes of the yeast Saccharomyces cerevisiae required for controlling sensitivity to 4-nitroquinoline-N-oxide and aminotriazole. Curr. Genet. 18: 93-96.

Gottesman, M. M. and Pastan, I. (1993) Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu. Rev. Biochem. 62: 385427.

Gottesman, M. M. and Pastan, I. (1997) Drug resistance: alterations in drug uptake or extrusion. In: Encyclopedia of Cancer, Vol. 1, pp. 549-559, Bertino, J. R. (ed.) Academic Press, San Diego, London, Boston, New York, Sydney, Tokyo and Toronto.

Gottesman, M. M., Currier, S., Bruggeman, E., Lelong, I., Stein, W. and Pastan, I. (1994) The multidrug transporter: mechanistic considerations. Curr. Top. Membr. Transp. 41: 3-17.

Gottesman, M. M., Hrycyna, C. A., Schoenlein, P. V., Germann, U. A. and Pastan, I. (1995) Genetic analysis of the multidrug transporter. Annu. Rev. Genet. 29: 607-609.

Grygorczyk, R. and Hanrahan, J. W. (1997a) Cystic fibrosis transmembrane conductance regulator and adenosine triphosphate: response. Science 275: 1325-1326.

Grygorczyk, R. and Hanrahan, J. W. (199713) CFTR-independent ATP release from epithelial cells triggered by mechanical stim- uli. Am. J. Physiol. 272 (Cell Physiol. 41): C1058-C1066.

Guiral, M., Viratelle, O., Westerhoff, H. V. and Lankelma, J.

(1994) Cooperative P-glycoprotein mediated daunorubicin transport into DNA-loaded plasma membrane vesicles. FEBS Lett. 346: 141-145.

Haase, E., Servos, J. and Brendel, M. (1992) Isolation and characterization of additional genes influencing resistance to various mutagens in the yeast Saccharomyces cereoisiae. Curr. Genet. 21: 319-324.

Hannun, Y. A. (1997) Apoptosis and the dilemma of cancer chemotherapy. Blood 89: 1845-1853.

Hayes, J. D. and Pulford, D. J. (1995) The glutathione S-transferase supergene family: regulation of GST* and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit. Rev. Biochem. Mol. Biol. 30: 445-600.

Hazen, K. C. (1995) New and emerging yeast pathogens. Clin. Microbial. Rev. 8: 462478.

Hedley, D. W., Xie, S. X., Minden, M. D., Choi, C. H., Chen, H.

and Ling, V. (1997) A novel energy dependent mechanism reducing daunorubicin accumulation in acute myeloid leukemia. Leukemia 11: 48-53.

Hegewisch-Becker, S. (1996) MDRl reversal: criteria for clinical trials designed to overcome the multidrug resistance phenotype. Leukemia 10 (Suppl. 3): S32-S38.

Heipieper, J. H., Weber, F. J., Sikkema, J., Keweloh, H. and de Bont, J. A. (1994) Mechanisms of resistance of whole cells to toxic organic solvents. Trends Biotechnol. 12: 409-415.

Hertle, K., Haase, E. and Brendel, M. (1991) The SNQ3 gene of Saccharomyces cereoisine confers hyper-resistance to several

functionally unrelated chemicals. Cum Genet. 19: 429-433. Higgins, C. F. (1994) Flip-flop: the transmembrane translocation

of lipids. Cell 79: 393-395.

Higgins, C. F. (1995) The ABC of channel regulation. Cell 82: 693-696.

Hindenburg, A. A., Gervasoni, J. E., Krishna, S., Stewart, V. J., Rosado, M., Lutzky, S., Bhalla, K., Baker, M. A. and Taub, R. N. (1989) Intracellular distribution and pharmacokinetics of daunorubicin in anthracycline-sensitive and -resistant HL-60 cells. Cancer Res. 49: 4607-4614.

Hirata, D., Yano, K., Miyahara, K. and Miyakawa, T. (1994) Sac- charomyces cerevisiae YDRI, which encodes a member of the

ATP-binding cassette (ABC) superfamily, is required for multidrug resistance. Curr. Genet. 26: 285-294.

Hollo, Z., Homolya, L., Davis, C. W. and Sarkadi, B. (1994) Cal-

cein accumulation as a fluorometric functional assay of the multidrug transporter. Biochim. Biophys. Acta 1191: 384-388.

Hollo, Z., Homolya, L., Hegedus, T. and Sarkadi, B. (1996) Trans- port properties of the multidrug resistance-associated protein (MRP) in human tumour cells. FEBS Lett. 383: 99-104.

Homolya, L., Hollo, Z., Germann, U. A., Pastan, I., Gottesman, M. M. and Sarkadi, B. (1993) Fluorescent cellular indicators are extruded by the multidrug resistance protein. J. Biol. Chem. 268: 21493-21496.

Houlbrook, S., Harris, A., Carmichael, J. and Stratford, 1. J. (1996) Relationship between topoisomerase 11 levels and resistance to topoisomerase II inhibitors in lung cancer cell lines. Anticancer Res. 16: 1603-1610.

Drug Resistance Mechanisms

Huehner, R. E. and Castro, K. G. (1995) The changing face of tuberculosis. Annu. Rev. Med. 46: 47-55.

Huovinen, I?., Sundstrom, L., Swedberg, G. and Skold, 0. (1995) Trimethoprim and sulfonamide resistance. Antimicrob. Agents Chemother. 39: 279-289.

Ishida, R., Hamatake, M., Wasserman, R. A., Nitiss, J. L., Wang, J. C. and Andoh, T. (1995) DNA topoisomerase II is the molecular target of bisdioxopiperazine derivatives ICRF- 159 and ICRF-193 in Saccharomyces cereuisiae. Cancer Res. 55: 2299-2303.

Ishikawa, T. (1992) The ATP-dependent glutathione S-conjugate export pump. Trends Biochem. Sci. 17: 463468.

Ishikawa, T., Wright, C. D. and Ishizuka, H. (1994) GS-X pump is functionally overexpressed in cis-diamminedichloroplatinum(II)-resistant human leukemia HL-60 cells and down-regulated by cell differentiation. J. Biol. Chem. 269: 29085-29093.

Ishikawa, T., Bao, J. J., Yamane, Y., Akimaru, K., Frindrich, K.,

Wright, C. D. and Kuo, M. T. (1996) Coordinated induction of MRP/GS X pump and gamma glutamylcysteine synthetase by

heavy metals in human leukemia cells. J. Biol. Chem. 271: 14981-14988.

Izquierdo, M. A., van der Zee, A. G., Vermorken, J. B., van der Valk, P., Belien, J. A., Giaccone, G., Scheffer, G. L., Flens, M. J., Pinedo, H. M., Kenemans, P., Meijer, C. J., Devries, E. G. and Scheper, R. J. (1995) Drug resistance associated marker LRP for prediction of response to chemotherapy and prognoses in advanced ovarian carcinoma. J. Natl. Cancer Inst. 87: 1230-1237.

Izquierdo, M. A., Scheffer, G. L., Flens, M. J., Giaccone, G., Brox- terman, H. J., Meijer, C. J., van der Valk, P. and Scheper, R. J. (1996a) Broad distribution of the multidrug resistance-related

vault lung resistance protein in normal human tissues and tumours. Am. J. Pathol. 148: 877-887.

Izquierdo, M. A., Shoemaker, R. H., Flens, M. J., Scheffer, G. L., Wu, L., Prather, T. R. and Scheper, R. J. (1996b) Overlapping phenotypes of multidrug resistance among panels of human cancer cell lines. Int. J. Cancer 65: 230-237.

Jedlitschky, G., Leier, I., Buchholz, U., Bamouin, K., Kurz, G. and Keppler, D. (1996) Transport of glutathione, glucuronate, and sulfate conjugates by the MRP gene encoded conjugate export pump. Cancer Res. 56: 988-994.

Jenkinson, H. F. (1996) Ins and outs of antimicrobial resistance:

era of the drug pumps. J. Dent. Res. 75: 736-742. Jentsch, T. J. and Gunther, W. (1997) Chloride channels: an

emerging molecular picture. Bioessays 19: 117-l 26.

Jilling, T. and Kirk, K. L. (1997) The biogenesis, traffic, and function of the cystic fibrosis transmembrane conductance regulator. Int. Rev. Cytol. 172: 193-241.

Josephhorne, T., Loeffler, R. S., Hollomon, D. W. and Kelly, S. L. (1996) Amphotericin B resistant isolates of Cryptococcus MO- formans without alteration in sterol biosynthesis. J. Med. Vet. Mycol. 34: 223-225.

Kaatz, G. W. and Seo, S. M. (1995) Inducible NorA-mediated multidrug resistance in Smphylococcus aurew. Antimicrob. Agents Chemother. 39: 2650-2655.

Kajiji, S., Talbot, F., Grizzuti, K., Van Dyke-Philips, V., Agresti, M., Safa, A. R. and Gros, P. (1993) Functional analysis of P-glycoprotein mutants identifies predicted transmembrane domain 11 as a putative drug binding site. Biochemistry 32:

41854194. Kanazawa, S., Driscoll, M. and Struhl, K. (1988) Saccharomyces

cerevisiue gene encoding a transmembrane protein required for aminotriazole resistance. Mol. Cell. BioI. 8: 664-673.

237

Kane, S. E. (1996) Multidrug resistance of cancer cells. Adv. Drug Res. 28: 181-252.

Karavokyros, I. and Delitheos, A. (1997) Effect of antineoplastic agents on non proliferating yeast cells: a possible membrane effect of doxorubicin. Anticancer Res. 17 (2A): 1079-1082.

Katzmann, D., Hallstrom, T. C., Voet, M., Wysock, W., Golin, J., Volckaert, G. and Moye-Rowley, W. S. (1995) Expression of an ATP-binding cassette transporter-encoding gene (YOR 1) is required for oligomycin resistance in Saccharomyces cerewisiae.

Mol. Cell. Biol. 15: 6875-6883. Kauh, E. A. and Bjornsti, M. A. (1995) SCTl mutants suppress

the camptothecin sensitivity of yeast cells expressing wild-type DNA topoisomerase I. Proc. Natl. Acad. Sci. USA 92: 6299- 6303.

Kavallaris, M., Madafiglio, J., Norris, M. D. and Haber, M. (1993)

Resistance to tetracycline, a hydrophilic antibiotic, is mediated by P-glycoprotein in human multidrug-resistant cells. Biochem. Biophys. Res. Commun. 190: 79-85.

Kaye, K. and Frieden, T. R. (1996) Tuberculosis control: the relevance of classic principles in an era of acquired immunodeficiency syndrome and multidrug resistance. Epidemiol. Rev. 18: 52-63.

Kelly, S. L., Lamb, D. C., Kelly, D. E., Manning, N. J., Loeffler, J.,

Hebart, H., Schumacher, U. and Einsele, H. (1997) Resistance to fluconazole and cross resistance to amphoterricin B in Cadida &cans from AIDS patients caused by defective sterol delta (5,6) desaturation. FEBS Lett. 400: 80-82.

Kivisto, K. T., Kroemer, H. K. and Eichelbaum, M. (1995) The

role of human cytochrome P-450 enzymes in the metabolism of anticancer agents: implications for drug interactions. Br. J. Clin. Pharmacol. 40: 523-530.

Kling, J. (1996) In vitro models for in vioo drug profiles. Nat. Bio- technol. 14: 1655-1656.

Klyachko, K. A., Schuldiner, S. and Neyfakh, A. A. (1997) Muta- tions affecting substrate specificity of the Bacillus sub&s multidrug transporter Bmr. J. Bacterial. 179: 2189-2193.

Kolaczkowski, M., van der Rest, M., Cybularz-Kolaczkowska, A., Soumillion, J. I’., Konings, W. N. and Goffeau, A. (1996) Anti- cancer drugs, ionophoric peptides, and steroids as substrates of the yeast multidrug transporter PdrSp. J. BioI. Chem. 271: 31543-31548.

Kono, M., O’Hara, K. and Ebisu, T. (1992) Purification and characterization of macrolide 2’0phosphotransferase type II from a strain of Escherichia coli highly resistant to macrolide antibiotics. FEMS Microbial. Lett. 76: 89-94.

Koomagi, R., Mattern, J. and Volm, M. (1995) Up-regulation of resistance-related proteins in human lung tumours with poor vascularization. Carcinogenesis 16: 2129-2133.

Kralli, A., Bohen, S. P. and Yamamoto, K. (1995) LEMl, an ATP- binding-cassette transporter, selectively modulates the biological potency of steroid hormones. Proc. Natl. Acad. Sci. USA 92: 47014705.

Krause, R. M. (1992) The origin of plagues: old and new. Science 257: 1073-1078.

Kremsner, P. G., Luty, A. J. and Graninger, W. (1997) Combina- tion chemotherapy for Plasmodium falciparum malaria. Parasitol. Today 13: 167-168.

Krogstad, D. J., Gluzman, I. Y., Kyle, D. E., Oduola, A. M., Mar- tin, S. K., Milhous, W. K. and Schlesinger, P. H. (1987) Efflux of chloroquine from Plasmodium falcipanrm: mechanism of chloroquine resistance. Science 238: 1283-1285.

Krogstad, D. J., Gluzman, I. Y., Herwaldt, B. L., Schlesinger, P. H. and Wellems, T. E. (1992) Energy dependence of chloroquine


accumulation and chloroquine efflux in Plasmodium fulciparum. Biochem. Pharmacol. 43: 5762.

Kuchler, K. and Thorner, J. (1992) Functional expression of human n&l cDNA in Saccharomyces cereoisiae. Proc. Natl. Acad. Sci. USA 89: 2302-2306.

Kuchler, K., Swartzman, E. E. and Thorner, J. (1994) A novel mechanism for transmembrane translocation of peptides: the Saccharomyces cerewisiae STE6 transporter and export of the mating pheromone a- factor. Curr. Top. Membr. Transp. 41: 19-42.

Kuge, S. and Jones, N. (1994) YAP1 dependent activation of TRX2 is essential for the response of Succharomyces cerevisiae to

oxidative stress by hydroperoxides. EMBO J. 13: 655-664. Kuge, S., Jones, N. and Nomoto, A. (1997) Regulation of YAP-1

nuclear localization in response to oxidative stress. EMBO J. 16:

1710-1720. Lautier, D., Canitrot, Y., Deeley, R. G. and Cole, S. P. (1996)

Multidrug resistance mediated by the multidrug resistance protein (MRP) gene. Biochem. Pharmacol. 52: 967-977.

Leclerq, R. and Courvalin, P. (1991) Bacterial resistance to macrolide, lincosamide, and streptogramin antibiotics by target modification. Antimicrob. Agents Chemother. 35: 1262-1272.

Lee, J. S., Paull, K., Alvarez, M., Hose, C., Monks, A., Grever, M., Fojo, A. T. and Bates, S. E. (1994) Rhodamine efflux patterns

predict P-glycoprotein substrates in the National Cancer Insti- tute drug screen. Mol. Pharmacol. 46: 627-638.

Leelapom, A., Paulsen, I. T., Tennent, J. M., Littlejohn, T. G. and

Skurray, R. A. (1994) Multidrug resistance to antiseptics and disinfectants in coagulase-negative staphylococci. J. Med. Microbial. 40: 214-220.

Leelaporn, A., Firth, N., Paulsen, I. T., Hettiaratchi, A. and Skur- ray, R. A. (1995) Multidrug resistance plasmid pSK108 from coagulase-negative staphylococci: relationships to Smphylococ- cus aureus qucC plasmids. Plasmid 34: 62-67.

Leier, I., Jedlitschky, G., Buchholz, U., Cole, S. P., Deeley, R. G. and Keppelr, D. (1994) The MRP gene encodes an ATP-dependent export pump for leukotriene C4 and related conjugates. J. Biol. Chem. 269: 27807-27810.

Leier, I., Jedlitschky, G., Buchholz, U., Center, M., Cole, S. P.,

Deeley, R. G. and Keppelr, D. (1996) ATP dependent glutathione disulphide transport mediated by the MRP gene encoded

conjugate export pump. Biochem. J. 314 (Part 2): 433-437. Leonard, J. P., Rathod, P. and Golin, J. (1994) Loss of function

mutation in the yeast multiple drug resistance gene PDR.5 causes a reduction in chloramphenicol efflux. Antimicrob. Agents Chemother. 38: 2492-2494.

Leppert, G., McDevitt, R., Falco, S. C., Van Dyk, T. K., Ficke, M. B. and Golin, J. (1990) Cloning by gene amplification of two loci conferring multiple drug resistance in Saccharomyces. Genetics 125: 13-20.

Li, X. Z., Livermore, D. M. and Nikaido, H. (1994) Role of efflux pump(s) in intrinsic resistance of Pseudomonas aeruginosa: resistance to tetracycline, chloramphenicol, and norfloxacin. Anti- microb. Agents Chemother. 38: 1732-1741.

Li, X. Z., Nikaido, H. and Poole, K. (1995) Role of MexA-MexB- OprM in antibiotic efflux in Pseudomonas aeruginosa. Antimi- crab. Agents Chemother. 39: 194881953.

Li, Z. E., Lu, Y. P., Zhen, R. G., Szczypka, M., Thiele, D. J. and Rea, P. A. (1997) A new pathway for vacuolar cadmium sequestration in Saccharomyces cereoisiae: YCFl -catalyzed transport of bis(glutathionato)cadmium. Proc. Natl. Acad. Sci. USA 94: 42-47.

Licht, T., Herrmann, F., Gottesman, M. M. and Pastan, I. (1997) In wioo drug selectable genes: a new concept in gene therapy. Stem Cells 15: 104-111.

Lind, U., Carlstedt-Duke, J., Gustafsson, J. A. and Wright, A. P. (1996) Identification of single amino acid substitutions of Cys- 736 that affect the steroid-binding affinity and specificity of the glucocorticoid receptor using phenotypic screening in yeast. Mol. Endocrinol. 10: 1358-1370.

Linden, P. K., Pasculle, A. W., Manez, R., Kramer, D. J., Fung, J. J., Pinna, A. D. and Kusne, S. (1996) Differences in outcomes for patients with bacteremia due to vancomycinresistant Entero- coccus faecium or vancomycin-susceptible E. faecium. Clin. Infect. Dis. 22: 663-670.

List, A. F., Spier, C. S., Grogan, T. M., Johnson, C., Roe, D. J., Greer,

J. P., Wolff, S. N., Broxtemran, H. J., Scheffer, G. L., Scheper, R. J. and Dalton, W. S. (1996) Overexpression of the major vault transporter protein lung resistance protein predicts treatment outcome in acute myeloid leukemia. Blood 87: 24642469.

Littlejohn, T. G., Paulsen, I. T., Gillespie, M. T., Tennent, J. M.,

Midgley, M., Jones, I. G., Purewal, A. S. and Skurray, R. A. (1992) Substrate specificity and energetics of antiseptic and dis- infectant resistance in Staphylococcus aureus. FEMS Microbial. Lett. 95: 259-266.

Livermore, D. M. (1995) B-Lactamases in laboratory and clinical resistance. Clin. Microbial. Rev. 35: 7-22.

Loe, D. W., Almquist, K. C., Cole, S. P. and Deeley, R. G. (1996a) ATP dependent 17 beta estradiol 17 (beta D glucuronide) transport by multidrug resistance protein (MRP): inhi-

bition by cholestatic steroids. J. Biol. Chem. 271: 9683-9689. Loe, D. W., Almquist, K. C., Deeley, R. G. and Cole, S. P.

(199613) Multidrug resistance protein (MRP) mediated transport of leukotriene C4 and chemotherapeutic agents in membrane vesicles: demonstration of glutathione dependent vincristine transport. J. Biol. Chem. 271: 9675-9682.

Longhurst, T. J., O’Neill, G. M. and Davey, R. A. (1996) The anthracycline resistance-associated (am) gene, a novel gene associated with multidrug resistance in a human leukaemia cell

line. Br. J. Cancer 74: 1331-1335. Mahato, S. B. and Garai, S. (1997) Advances in microbial steroid

biotransformation. Steroids 62: 332-345.

Mahe, Y., Lemoine, Y. and Kuchler, K. (1996a) The ATP binding cassette transporters Pdr5 and Snq2 of Saccharomyces cereoisiae can mediate transport of steroids in vioo. J. Biol. Chem. 271: 25167-25172.

Mahe, Y., Parle-McDermott, A., Nourani, A., Delahodde, A., Lamprecht, A. and Kuchler, K. (1996b) The ATP-binding cassette multidrug transporter Snq2p of Sacchromyces cereoisiae: a novel target for the transcription factors Pdrl and Pdd. Mol. Microbial. 20: 109-l 17.

Marichal, P. and Vanden Bossche, H. (1995) Mechanisms of resistance to azole antifungals. Acta Biochim. Pol. 42: 509-516.

Markham, P. N. and Neyfakh, A. A. (1996) Inhibition of the multidrug transporter NorA prevents emergence of norfloxacin

resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 40: 2673-2674.

Martinez, L., Hemandezalles, S., Alberti, S., Tomas, J. M., Benedi, V. J. and Jacoby, G. A. (1996) In viva selection of porin deficient mutants of Klebsiellu pneumoniae with increased resistance to cefoxitin and expanded spectrum cephalosporins. Antimi- crab. Agents Chemother. 40: 342-348.

Mashima, T., Naito, M., Kataoka, S. and Tsuruo, T. (1996) Cancer chemotherapy and apoptosis. Nippon Rinsho 54: 1935-1942.


Mattem, J. and Volm, M. (1993) Multiple pathway drug resistance (Review). Int. J. Oncol. 2: 557-561.

McCall, K. and Steller, H. (1997) Facing death in the fly: genetic analysis of apoptosis in Drosophila. Trends Genet. 13: 222-226.

McMurry, L., Petrucci, R. E., Jr. and Levy, S. B. (1980) Active efflux of tetracycline encoded by four genetically different tetracycline resistance determinants in Escherichia coli. Proc. Natl.

Acad. Sci. USA 77: 39743977. Meyers, S., Schauer, W., Balzi, E., Wagner, M., Goffeau, A. and

Golin, J. (1992) Interaction of the yeast pleiotropic drug resis-

tance genes PDR 1 and PDR5. Curr. Genet. 2 1: 43 l-436. Miyahara, K., Hirata, D. and Miyakawa, T. (1996a) YAP-l- and

YAP-2-mediated, heat shock-induced transcriptional activation of the multidrug resistance ABC transporter genes in Sac-

charomyces cerevisiae. Curr. Genet. 29: 103-105. Miyahara, K., Mizunuma, M., Hirata, D., Tsuchiya, E. and Miya-

kawa, T. ( 199613) The involvement of the Sacchomyces cereui-

siae multidrug resistance transporters Pdr5p and Snq2p in cation resistance. FEBS Lett. 399: 317-320.

Molla, A., Korneyeva, M., Gao, Q., Vasavanonda, S., Schipper, P. J., MO, H. M., Markowitz, M., Chemyavskiy, T., Niu, P., Lyons, N., Hsu, A., Granneman, G. R., Ho, D. D., Boucher, C.

A., Leonard, J. M., Norbeck, D. W. and Kempf, D. J. (1996) Ordered accumulation of mutations in HIV protease confers

resistance to ritonavir. Nat. Med. 2: 760-766. Moritz, T. and Williams, D. A. (1997) Transfer of drug resistance

genes to hematopoietic precursors. In: Encyclopedia of Cancer, Vol. 3, pp. 549-559, Bertino, J. R. (ed.) Academic Press, San Diego, London, Boston, New York, Sydney, Tokyo, Toronto.

Morris, J. G., Jr., Shay, D. K., Hebden, J. N., MC Carter, R. J., Jr., Perdue, B. E., Jarvis, W., Johnson, J. A., Dowling, T. C., Polish, L. B. and Schwalbe, R. S. (1995) Enterococci resistant to multiple antimicrobial agents including vancomycin. Ann. Intern.

Med. 123: 250-259. Morris, S., Bai, G. H., Suffys, P., Portillo-Gomez, L., Fairchok, M.

and Rouse, D. (1995) Molecular mechanisms of multiple drug resistance in clinical isolates of Mycobacterium tuberculosis. J.

Infect. Dis. 171: 954-960. Moye-Rowley, W. S., Harshmann, K. D. and Parker, C. S. (1989)

Yeast YAP1 encodes a novel form of the Jun family of transcriptional activator protein. Genes Dev. 3: 283-292.

Muller, M., Meijer, C., Zaman, G. J., Borst, P., Schepper, R. J., Mul- der, N. H., Devries, E. G. E. and Jansen, P. L. (1994) Overexpres- sion of the gene encoding the multidrug resistance associated

protein results in increased ATP dependent glutathione S conjugate transport. Proc. Natl. Acad. Sci. USA 91: 13033-13037.

Nakae, T. (1995) Role of membrane permeability in determining antibiotic resistance in Pseudomonas ueruginosa. Microbial. Immunol. 39: 221-229.

Nelissen, B., Mordant, P., Jonniaux, J. L., De Wachter, R. and

Goffeau, A. (1995) Phylogenetic classification of the major superfamily of membrane transport facilitators, as deduced from yeast genome sequencing. FEBS Lett. 377: 232-236.

Neu, H. C. (1992) The crisis in antibiotic resistance. Science 257:

1064-1073. Nicolas, P. and Mor, A. (1995) Peptides as weapons against micro-

organisms in the chemical defence system of vertebrates. Annu. Rev. Microbial. 49: 277-304.

Nikaido, H. (1994) Prevention of drug access to bacterial targets: permeability barriers and active efflux. Science 264: 382-388.

Nitiss, J. L. (1994) Using yeast to study resistance to topoisomerase R-targeting drugs. Cancer Chemother. Pharmacol. 34: S6-Sl3.

Nitiss, J. and Beck, W. T. (1996) Antitopoisomerase drug action and resistance. Eur. J. Cancer 32A: 958-966.

Nitiss, J, and Wang, J. C. (1988) DNA topoisomerase-targeting antitumor drugs can be studied in yeast. Proc. Natl. Acad. Sci. USA 85: 7501-7505.

Odds, F. C. (1996) Resistance of clinically important yeasts to antifungal agents. Int. J. Antimicrob. Agents 6: 145-147.

Ortiz, D. F., Pierre, M. V., Abdulmessih, A. and Arias, I. M. (1997) A yeast ATP-binding cassette-type protein mediating ATP-dependent bile acid transport. J. Biol. Chem. 272: 15358-

15365. Ouabdesselam, S., Tankovic, J. and Soussy, C. J. (1996) Qui-

nolone resistance mutations in the gyrA gene of clinical isolates of Salmonella. Microb. Drug Resist. 2: 299-302.

Oude Elferink, R. P., Tytgat, G. N. and Groen, A. K. (1997) The role of mdr2 P-glycoprotein in hepatobiliary lipid transport. FASEB J. 11: 19-28.

Ouelette, M. and Papadopoulou, B. (1993) Mechanisms of drug resistance in Leishmania. Parasitol. Today 9: 150-153.

Papadopoulou, B., Roy, G., Dey, S., Rosen, B. P. and Ouelette, M. (1994) Contribution of the Leishmania P-glycoprotein-related gene ltp@A to oxyanion resistance. J. Biol. Chem. 269: 11980- 11986.

Paul, S., Belinsky, M. G., Shen, H. X. and Kruh, G. D. (1996a)

Structure and in vitro substrate specificity of the murine multidrug resistance associated protein. Biochemistry 35: 13647- 13655.

Paul, S., Breuninger, L. M. and Kruh, G. D. (1996b) ATP dependent transport of lipohilic cytotoxic drugs by membrane vesicles

prepared from MRP overexpressing HL60/ADR cells. Biochem- istry 35: 14003-14011.

Paul, S., Breuninger, L. M., Tew, K. D., Shen, H. X. and Kruh, G. D. (1996~) ATP dependent uptake of natural product cytotoxic drugs by membrane vesicles establishes MRP as a broad

specificity transporter. Proc. Natl. Acad. Sci. USA 93: 6929- 6934.

Paulsen, I. T., Brown, M. H. and Skurray, R. (1996a) Proton-depen-

dent multidrug eftlux systems. Microbial. Rev. 60: 575-608. Paulsen, I. T., Skurray, R. A., Tam, R., Saler, M. H., Turner, R. J.,

Weiner, J. H., Goldberg, E. B. and Grinius, L. L. (1996b) The SMR family: a novel family of multidrug efflux proteins involved with the efflux of lipophilic drugs. Mol. Microbial. 19: 1167-1175.

Paulusma, C. C., Bosma, P. J., Zaman, J. R., Bakker, C. T., Otter, M., Scheffer, G. L., Scheper, R. J., Borst, P. and Oude-Elferink, R. P. (1996) Congenital jaundice in rats with a mutation in a multidrug resistance-associated protein gene. Science 271: 1126-1128.

Pawagi, A. B., Wang, J., Silverman, M., Reithmeier, R. A. and

Deber, C. M. (1994) Transmembrane amino acid distribution in P-glycoprotein: a functional role in broad substrate specificity. J. Mol. Biol. 235: 554-564.

Pompon, D., Perret, A., Bellamine, A., Laine, R., Gautier, J. C.

and Urban, P. (1995) Genetically engineered yeast cells and their applications. Toxicol. Lett. 82: 8155822.

Poole, K. (1994) Bacterial multidrug resistance: emphasis on efflux mechanisms and Psewlomonas aeruginosa. J. Antimicrob. Chemo- ther. 34: 453-456.

Prasad, R., de Wergifosse, P., Goffeau, A. and Balzi, E. (1995) Molecular cloning and characterization of a novel gene of Can- dida albicuns, CDRI , conferring multiple resistance to drugs and antifungals. Curr. Genet. 27: 320-329.


Price, M. F., LaRocco, M. T. and Gentry, L. 0. (1994) Flucon- azole susceptibilities of Candida species and distribution of spe-

cies recovered from blood cultures over a 5-year period. Antimicrob. Agents Chemother. 38: 1422-1427.

Pussard, E. and Verdier, F. (1994) Antimalarial 4.aminoquinolo-

nes: mode of action and pharmacokinetics. Fund. Clin. Phar-

macol. 8: 1-7.

Raviv, Y., Pollard, H. B., Bruggemann, E. P., Pastan, I. and Got-

tesman, M. M. (1990) Photosensitized labeling of a functional

multidrug transporter in living drug-resistant tumor cells. J.

Biol. Chem. 265: 3975-3980.

Raymond, M., Gros, I’., Whiteway, M. and Thomas, D. Y. (1992)

Functional complementation of yeast ste6 by a mammalian

multidrug resistance mdr gene. Science 256: 232-234. Reddy, M. M., Quinton, P. M., Haws, C., Wine, J. J., Grygorczyk, R.,

Tabcharani, J. A., Hanrahan, J. W., Gunderson, K. L. and Kopito,

R. R. (1997) Cystic fibrosis transmembrane conductance regula-

tor and adenosine triphosphate: response. Science 275: 1325.

Reed, J. C., Miyashita, T., Takayama, S., Wang, H. G., Sato, T.,

Krajewski, S., Aimesempe, C., Bodrug, S., Kitada, S. and Han-

ada, M. (1996) Bcl-2 family proteins: regulators of cell death

involved in the pathogenesis of cancer and resistance to ther-

apy. J. Cell. Biochem. 60: 23-32.

Reid, R. J., Kauh, E. A. and Bjornsti, M. A. (1997) Camptothecin

sensitivity is mediated by the pleiotropic drug resistance net-

work in yeast. J. Biol. Chem. 272: 12091-12099.

Roberts, M. C. (1994) Epidemiology of tetracycline-resistance

determinants. Trends Microbial. 2: 353-357.

Roepe, P. D. (1997) Biophysical aspects of P-glycoprotein medi-

ated multidrug resistance. Int. Rev. Cytol. 171: 121-165.

Roessner, C. A. and Scott, A. I. (1996) Genetically engineered

synthesis of natural products: from alkaloids to corrins. Annu.

Rev. Microbial. 50: 467-490.

Rosenberg, M. F., Callaghan, R., Ford, R. C. and Higgins, C. F.

(1997) Structure of the multidrug resistance P-glycoprotein to

2.5 nm resolution determined by electron microscopy and

image analysis. J. Biol. Chem. 272: 10685-10694.

Ross, J. I., Eady, E. A., Cove, J. H., Cunliffe, W. J., Baumberg, S.

and Wootton, J. C. (1990) Inducible erythromycin resistance in

staphylococci is induced by a member of the ATP-binding

transport super-gene family. Mol. Microbial. 7: 1207-1214.

Rubio, J. P. and Cowman, A. F. (1996) The ATP-binding cassette

(ABC) gene family of Plasmodium fakiparum. Parasitol. Today

12: 135-140.

Ruetz, S. and Gros, P. (1994a) Functional expression of P-glycopro-

teins in secretory vesicles. J. Biol. Chem. 269: 12277-12284.

Ruetz, S. and Gras, P. (1994b) A mechanism of P-glycoprotein

action in multidrug resistance: are we there yet? Trends Phar-

macol. Sci. 15: 260-263.

Ruetz, S., Brault, M., Kast, C., Hemenway, C., Heitman, J., Grant, C. E., Cole, S. P., Deeley, R. G. and Gras, P. (1996) Functional expression of the multidrug resistance associated protein in the

yeast Succharomyces cereuisiae. J. Biol. Chem. 271: 4154-4160.

Samuleson, J., Ayala, P., Orozco, E. and Wirth, D. (1990) Emetine-

resistant mutants of Entamoebu histolytica overexpress mRNAs for multidrug resistance. Mol. Biochem. Parasitol. 66: 161-164.

Sancar, A. (1995) DNA repair in humans. Annu. Rev. Genet. 29: 69-105.

Sanchez, C. P., Wunsch, S. and Lanzer, M. (1997) Identification of a chloroquine importer in Plasmodium fakiparum: differences in import kinetics are genetically linked with the chloroquine- resistant phenotype. J. Biol. Chem. 272: 2652-2658.

Sanglard, D., Kuchler, K., Ischer, F., Pagani, J. L., Monod, M. and Bille, J. (1995) Mechanisms of resistance to azole antifungal

agents in Candida a&cans isolates from AIDS patients involve specific multidrug transporters. Antimicrob. Agents Chemother.

39: 2378-2386.

Sanglard, D., Ischer, F., Monod, M. and Bille, J. (1996) Suscepti-

bilities of Candida albicans multidrug transporter mutants to var-

ious antifungal agents and other metabolic inhibitors.

Antimicrob. Agents Chemother. 40: 2300-2305.

Sanglard, D., Ischer, F., Monod, M. and Bille, J. (1997) Cloning of

Candid0 albican.5 genes conferring resistance to azole antifungal agents: characterization of CDRZ, a new multidrug ABC trans-

porter gene. Microbiology 143: 405-416. Saxena, M., Singhal, S. S., Awasthi, S., Singh, S. V., Labelle, E. F.,

Zimniak, P. and Awasthi, Y. C. (1992) Dinitrophenyl S-glu-

tathione ATPase purified from human muscle catalyzes ATP

hydrolysis in the presence of leukotrienes. Arch. Biochem. Bio-

phys. 298: 231-237.

Scheffer, G. L., Wijngaard, P. L., Flens, M. J., Izquierdo, M. A.,

Slovak, M. L., Pinedo, H. M., Meijer, C. J., Clevers, H. C. and

Scheper, R. J. (1995) The drug resistance related protein LRP is

a major vault protein. Nat. Med. 1: 578-582.

Schmit, J. C., Cogniaux, J., Hermans, P., Van Vaeck, C., Spre-

cher, S., Van Remoortel, B., Witvrouw, M., Balzarini, J.,

Desmyter, J., De Clerq, E. and Vandamme, A. M. (1996) Multi-

ple drug resistance to nucleoside analogues and nonnucleoside

reverse transcriptase inhibitors m an efficiently replicating

human immunodeficiency virus type 1 patient strain. J. Infect.

Dis. 174: 962-968.

Schnappinger, D. and Hillen, W. (1996) Tetracyclines: antibiotic

action, uptake, and resistance mechanisms. Arch. Microbial.

165: 359-369.

Schnell, N., Krems, B. and Entian, K. D. (1992) The PAR1

(YAPl/SNQ3) gene of Sacchuromyces cereoisiae, a c-jun homo-

logue, is involved in oxygen metabolism. Curr. Genet. 21: 269-

273.

Schuldiner, S., Shirvan, A. and Linial, M. (1995) Vesicular neu-

rotransmitter transporters: from bacteria to humans. Physiol. Rev. 75: 369-392.

Schuldiner, S., Lebendiker, M. and Yerushalmi, H. (1997) EmrE,

the smallest ion-coupled transporter, provides a unique para-

digm for structure-function studies. J. Exp. Biol. 200: 335-341.

Seimiya, H., Mashima, T., Toho, M. and Tsuruo, T. (1997) c-Jun

NHz-terminal kinase-mediated activation of interleukin-I@

converting enzyme/CED-3-like protease during anticancer

drug-induced apoptosis. J. Biol. Chem. 272: 4631-4636.

Senior, A. E., Al-Shawi, M. K. and Urbatsch, I. L. (1995) The cat-

alytic cycle of P-glycoprotein. FEBS Lett. 377: 285-289.

Service, R. F. (1995) Antibiotics that resist resistance. Science

270: 724-727. Servos, J., Haase, E. and Brendel, M. (1993) Gene ShrQ2 of Sac-

charomyces cereuisti, which confers resistance to 4-nitroquino-

line-N-oxide and other chemicals, encodes a 169 kDa protein

homologous to ATP-dependent permeases. Mol. Gen. Genet. 236: 214-218.

Shapiro, A. B. and Ling, V. (1995) Reconstitution of drug trans-

port by purified P-glycoprotein. J. Biol. Chem. 270: 16167- 16175.

Shaw, K. J., Rather, P. N., Hare, R. S. and Miller, G. H. (1993) Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes. Microbial. Rev. 57: 138-163.


Shaw, W. V. and Leslie, A. G. (1989) Chloramphenicol acetyl

transferases. In: Handbook of Experimental Pharmacology:

Microbial Resistance to Drugs, Vol. 91, pp. 313-324, Bock, A.

and Bryan, L. E. (eds.) Springer-Verlag, Berlin. Shen, H. X., Paul, S., Breuninger, L. M., Ciaccio, P. J., Laing,

N. M., Helt, M., Tew, K. D. and Kruh, G. D. (1996) Cellular and in vitro transport of glutathione conjugates by MRP. Bio-

chemistry 35: 5719-5725. Simon, S. M. and Schindler, M. (1994) Cell biological mecha-

nisms of multidrug resistance in tumors. Proc. Natl. Acad. Sci.

USA 91: 3497-3504. Skovsgaard, T., Nielsen, D., Maare, C. and Wassermann, K.

(1994) Cellular resistance to cancer chemotherapy. Int. Rev.

Cytol. 156: 77-157. Song, M. D., Wachi, M., Doi, M., Ishino, F. and Matsuhashi, M.

(1987) Evolution of an inducible penicillin-target protein in

methicillin-resistant Staphylococcus aureu~ by gene fusion. FEBS Lett. 221: 167-171.

Spratt, B. G. (1994) Resistance to antibiotics mediated by target alterations. Science 264: 388-393.

Stahl, S. and Uhlen, M. (1997) Bacterial surface display: trends and progress. Trends Biotechnol. 15: 185-192.

Steller, H. (1995) Mechanisms and genes of cellular suicide. Sci-

ence 267: 1445-1449. St-Pierre, M. V., Ruetz, S., Epstein, L. F., Gros, I?. and Arias, I. M.

(1994) ATP-dependent transport of organic anions in secretory vesicles of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA

91: 9476-9479. Suarez, J. E. and Mendoza, M. C. (1991) P&mid-encoded fosfomy-

tin resistance. Antimicrob. Agents Chemother. 35: 791-795.

Taguchi, Y., Yoshida, A., Takada, Y., Komano, T. and Ueda, K. (1997) Anticancer drugs and glutathione stimulate vanadate induced trapping of nucleotide in multidrug resistance associated protein (MRP). FEBS Lett. 401: 11-14.

Taiz, L. and Zeiger, E. (1991) Surface protection and secondary defense compounds. In: Plant Physiology, pp. 318-345, Taiz, L. and Zeiger, E. (eds.) The Benjamin/Cummings, Redwood City, Menlo Park, Reading, New York, Don Mills, Wokingham, Amsterdam, Bonn, Sydney, Singapore, Tokyo, Madrid, San Juan.

Takiff, H. E., Cimino, M., Musso, M. C., Weisbrod, T., Martinez, R., Delgado, M. B., Salazar, L., Bloom, B. R. and Jacobs, W. R.,

Jr. (1996) Efflux pump of the proton antiporter family confers low-level fluoroquinolone resistance in Mycobucrerium smegma- tis. Proc. Natl. Acad. Sci. USA 93: 362-366.

Thanassi, D. G., Suh, G. S. and Nikaido, H. (1995) Role of outer membrane barrier in efflux mediated tetracycline resistance of Escherichia cob. J. Bacterial. 177: 998-1007.

Thompson, C. B. (1995) Apoptosis in the pathogenesis and treatment of disease. Science 267: 1456-1462.

Travis, J. (1994) Reviving the antibiotic miracle. Science 264:

360-361. Tuite, M. F. (1996) Discovery and development of new systemic

antifungals. Trends Biotechnol. 14: 219-220. Ullman, B. (1995) Multidrug resistance and P-glycoproteins in

parasitic protozoa. J. Bioenerg. Biomembr. 27: 77-84. Vanden Bossche, H. (1995) Chemotherapy of human fungal

infections. In: Modern Selective Fungicides: Properties, Appli-

cations, Mechanism of Action, pp. 431484, Lyr, H. (ed.) Gustav Fischer Verlag, Jena.

Vanden Bossche, H., Marichal, P. and Odds, F. C. (1994) Molecu- lar mechanisms of drug resistance in fungi. Trends Microbial. 2: 393-400.

Van Es, H. H., Karcz, S., Chu, F., Cowman, A. F., Vidal, S., Gras,

P. and Schurr, E. (1994a) Expression of the plasmodial pfmdrl gene in mammalian cells is associated with increased suscepti-

bility to chloroquine. Mol. Cell. Biol. 14: 2419-2428. Van Es, H. H., Renkema, H., Aerts, H. and Schurr, E. (1994b)

Enhanced lysosomal acidification leads to increased chloroquine accumulation in CHO cells expressing the pfmdrl gene. Mol. B&hem. Parasitol. 68: 209-219.

Van Helvoort, A., Smith, A. J., Sprong, H., Fritzsche, I., Schinkel, A. H., Borst, P. and van Meer, G. (1996) MDRl P-glycoprotein is a lipid translocase of broad specificity, while MDR3 P-glycoprotein specifically translocates phosphatidylcholine. Cell 87: 507-515.

Van Veen, H. W., Venema, K., Bolhuis, H., Oussenko, I., Kok, J., Poolman, B., Driessen, A. J. and Konings, W. N. (1996) Multi- drug resistance mediated by a bacterial analog of the human

drug transporter MDRl. Proc. Natl. Acad. Sci. USA 93: 10668-10672.

Voishvillo, N. E., Turuta, A. M. and Kamernitsky, A. V. (1994) Microorganisms as reagents for transformations of 5a-steroids. Russ. Chem. Bull. 43: 567-588.

Volkman, S. K., Cowman, A. F. and Wirth, D. F. (1996) Func- tional complementation of the ste6 gene of Saccharomyces cerwisiae with the pfmdrl gene of Plasmodium fakipurum. Proc. NatI. Acad. Sci. USA 93: 3479-3491.

Wackett, L. P. (1997) Bacterial biocatalysis: stealing a page from nature’s book. Nat. Biotechnol. 15: 415-416.

Walsh, C. T. (1993) Vancomycin resistance: decoding the molec-

ular logic. Science 261: 3088309. Erratum: Science 262: 164

(1993). Ward, 0. P. and Young, C. S. (1990) Reductive biotransforma-

tions of organic compounds by cells or enzymes of yeast. Enzyme Microb. Technol. 12: 482-493.

Wei, L. Y., Stutts, M. J., Hoffman, M. M. and Roepe, P. D. (1995) Overexpression of the cystic fibrosis transmembrane conductance regulator in NIH 3T3 cells lowers membrane potential and intracellular pH and confers a multidrug resistance phenotype. Biophys. J. 69: 883-895.

Weisburg, J. H., Curcio, M., Caron, P. C., Raghu, G., Mechetner, E. B., Roepe, P. D. and Scheinberg, D. A. (1996) The multidrug resistance phenotype confers immunological resistance. J. Exp. Med. 183: 2699-2704.

Wellems, T. E., Panton, J., Gluzman, I. Y., do Rosario, V. E., Gwadz, R. W., Walker-Jonah, A. and Krogstad, D. J. (1990) Chloroquine resistance not linked to n&-like genes in a Plas- modium falciparum cross. Nature 345: 253-255.

Wellems, T. E., Walker-Jonah, A. and Panton, L. J. (1991) Genetic mapping of the chloroquine resistance locus on Plas- modium fukipurum chromosome 7. Proc. Natl. Acad. Sci. USA 88: 3382-3386.

Wemmie, J. A., Szczypka, M. S., Thiele, D. J. and Moye-Rowley, W. S. (1994) Cadmium tolerance mediated by the yeast AI’-1 protein requires the presence of an ATP-binding cassette transporter-encoding gene, YCFl J. Biol. Chem. 269: 32592- 32597.

Williams, J. B. (1996) Drug efflux as a mechanism of resistance. Br. J. Biomed. Sci. 53: 290-293.

Wilson, C. M., Serrano, A. E., Wasley, A., Bogenschutz, M. P., Shankar, A. H. and Wirth, D. F. (1989) Amplification of a

gene related to mammalian mdr genes in drug-resistant falciparum malaria. Science 244: 1184-l 186.

Wilson, C. M., Volkman, S. K., Thaithong, S., Martin, R. K.,


Kyle, D. E., Milhous, W. K. and Wirth, D. F. (1993) Amplifica- tion of pfmdrl associated with mefloquine and halofantrine resistance in PIasmodium jnlcipurum from Thailand. Mol. Bio- them. Parasitol. 57: 151-160.

Wooden, J. M., Hartwell, L. H., Vasquez, B. and Sibley, C. H. (1997) Analysis in yeast of antimalaria drugs that target the dihydrofolate reductase of Plasmodium falcigarum. Mol. Bio- them. Parasitol. 85: 25-40.

Wu, A., Wemmie, J. A., Edgington, N. P., Goebl, M., Guevara, J.

L. and Moye-Rowley, W. S. (1993) Yeast bZip proteins mediate pleiotropic drug resistance and metal resistance. J. Biol. Chem. 268: 18850-18858.

Yelin, R. and Schuldiner, S. (1995) The pharmacological profile

of the vesicular monoamine transporter resembles that of multidrug transporters. FEBS Lett. 377: 201-207.

Zachowski, A. (1993) Phospholipids in animal eukaryotic membranes: transverse asymmetry and movement. Biochem. J. 294:

1-14. Zaman, G. J., Cnubben, N. H., Vanbladeren, P. J., Evers, R. and

Borst, P. (1996) Transport of glutathione conjugate of ethacrynic acid by the human multidrug resistance protein MRP. FEBS Lett. 391: 126-130.

Zamble, D. B. and Lippard, S. J. (1995) Cisplatin and DNA repair in cancer chemotherapy. Trends Biochem. Sci. 20: 435439.

Zielenski, J. and Tsui, L. C. (1995) Cystic fibrosis: genotypic and

phenotypic variations. Annu. Rev. Genet. 29: 777-807.

active efflux by multidrug transporters as one of the strategies

Documents

advanced ovarian

acquired immunodeficiency

proton motive

acute myeloid

van der valk

kralli ec

major facilitator

altered specificity