P-glycoprotein-mediated multidrug resistance in tumor cells: Biochemistry, clinical relevance and modulation

Molec. Aspects Med. Vol. 16, pp. l-78, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved. 0096-2997195 $29.00

Pergamon

0098-2997(94)00040-9

P-Glycoprotein-Mediated Multidrug Resistance in Tumor Cells: Biochemistry, Clinical Relevance and Modulation Chaim Shustik”, William Daltonf’ and Philippe Gros$ Departnrent

of Medicine and McGill Cancer Center, Royal Victoria Hospital and McGill University, Montreal, Quebec, Canada tArizona Cancer Center and University of Arizona, Tucson, Arizona, U.S.A. #Department of Biochemistry, McGill University, Montreal, Quebec, Canada

Contents

PREFACE CHAPTEiR 1

3 Structural and Functional Aspects Role in Multidrug Resistance

CHAPTEiR 2

Multidrug

CHAPTEiR 3

Modulation

Resistance

of P-Glycoprotein

and its 5

in the Clinic

21 43

of P-Glycoprotein

53

REFERENCES

All correslpondence should be addressed to: Philippe Gros, Department 3655 Drummond St., Montreal, Quebec, Canada, H3G-lY6.

1

of Biochemistry,

McGill University,

Preface

A major limitation to the successful chemotherapeutic treatment of cancer is the natural or acquired resistance of tumor cells to cytotoxic drugs. While some types of solid tumors s,uch as lung, kidney and pancreas intrinsically respond very poorly to drug treatment, others, most notably leukemias and lymphomas, only become resistant to treatment upon relapse after an initial response. The latter phenomenon is most intriguing as tumor cells can acquire in vivo simultaneous resistance to structurally unrelated compounds to which they have never been exposed to previously. This phenomenon, known as multidrug resistance (MDR), involves a wide range of natural products including Vinca alkaloids and anthracyclines and has been the focus of intense .investigation over the past twenty years. Dramatic progress has been made recently in the understanding of the molecular basis of this phenomenon, including the identification of the proteins involved, the genes that encode them and the mechanism by which these proteins operate. This research has allowed the production of diagnostic reagents such as antibodies and specific nucleic acid probes that monitor, and in some instances, predict the emergence of multidrug resistance both in vitro and in vivo. In addition, the biochemical and genetic dissection of the protein (P-glycoprotein) and gene (MDR) involved in MDR has allowed a further understanding of the mechamsm of action of P-glycoprotein, and has provided in vitro and in vivo model systems for the rational design and testing of new drugs or modulators capable of by-passing or blocking its action. In this review, we will summarize the current status of knowledge on three major aspects of P-glycoprotein-mediated multidrug resistance: (1) the structure-function analysis of P-glycoproteins and the mechanism by which they confer MDR; (2) the clinical data on the role of P-glycoprotein in resistance to chemotherapy in vivo in human tumors, and (3) the clinical evaluation of agents capable of blocking the action of this protein. Since the published information on the topic of resistance to chemotherapy in tumors is vast, this review is necessarily selective. In particular, several important additional aspects of MDR will only be briefly reviewed; these include the inventory or biochemical analysis of the many multidrug resistant cell lines that have been used to study P-glyco:protein, the physiological aspects of drug transport and the pharmacology of the dru,gs involved. These topics have been discussed recently in excellent reviews and books to which the reader can refer to (Pinedo et al., 1992; Roninson, 1991). Finally, we will restrict our review to the P-glycoprotein-mediated MDR and will not discuss the equally important non-P-glycoprotein-mediated mechanisms or drug resistance. 3

Chapter 1

Structural and Functional Aspects of P-Glycoprotein Multidrug Resistance

and its Rote in:

Characteristics of the Multidrug Resistance Phenotype In Vitro The stud.y of multidrug resistance and the characterization of cellular changes associated with it, have been inherently difficult to perform in vivo in clinical specimens. The difficulty in obtaining serial samples from the same tumor during the course of treatment, the heterogeneous nature of the tumor cell populations, and the low levels of resistance required to achieve clinical resistance in vivo are but a few factors that preclude a systematic analysis in vivo. Therefore, elucidation of the genetic, biochemical and pharmacological bases of this phenomenon have relied almost exclusively on the availability of in vitro tissue culture models. These have proven to be easy to generate and highly informative. Multidrug-resistant cell lines have been obtained traditionally by empirical protocols using step-wise selection in culture medium containing a single cytotoxi’c agent (Biedler and Riehm, 1970; Beth-Hansen et al., 1976; Fojo et al., 1985a; Lemontt et al., 1988). In this type of experiment, large numbers of drug-sensitive cells are plated in medium containing low concentrations of a single drug, leading to the low frequency appearance of drug-resistant colonies several weeks later. These cells are then expanded and the exercise is repeated several times in increasing concentration of drug. Over the years, a large number of independently-derived and highly resistant human and rodent cell lines from different tissue origins have been derived successfully using a number of cytotoxic drugs (reviewed by Beck and Danks, 1991; Sugimoto and Tsuruo, 1991). One of the most remarkable findings of early studies on these cell lines was that their phenotypic characteristics were strikingly similar despite the diversity of their tissue origin, in vitro selection procedures and drugs used in their production, suggesting that a common mechanism underlay the emergence of resistance. (For comprehensive reviews, see Endicott and Ling, 1989; Roninson, 1991, Gottesman and Pastan, 1993.) First, these MDR cell lines display a very similar profile of cross-resistance to structurally and functionally unrelated cytotoxic drugs, although resistance is sometimes, but not always highest for the drug used in the initial selection. This group of drugs (known as the MDR type of drugs) includes Vinca alkaloids (vinblastine, vincristine), anthracyclines (adriamycin), colchicine, actinomycin D, etoposides (VP-16, VM-21), topoisomerase inhibitors (amsacrine), taxol and many others, including small peptides such as, valinomycin and gramicidin D (e.g. see Gupta, 1983; Tsuruo et al., 1983; 5

6

C. Shustik et al.

Conter and Beck, 1984). There are very few structural or functional characteristics that are common amongst MDR drugs: they are all very hydrophobic molecules that enter the cell by passive diffusion across the membrane-lipid bilayer. In general, they are biplanar, amphiphilic and usually contain a basic nitrogen atom bearing a positive charge at neutral pH (Zamora et al., 1988; Pearce et al., 1989). Since the identification of the structural determinants on the drug molecule defining the ‘MDR phenotype’ is an important prerequisite for the design of alternative and perhaps more effective drugs; much effort has been directed towards the characterization of such determinants. A comprehensive study of derivatives of the anthracyclines ellipticine and olivacine indicated that lipid solubility, the presence of a basic nitrogen atom and at least two aromatic rings were essential requirements (Chevallier-Multon et al., 1990). More detailed studies of analogs of another drug, colchicine, revealed that not only the chemical composition of the molecule, but also its size were important (Tang-Wai et al., 1993). Colchicine is a plant alkaloid composed of three rings, two of which are aromatic and form the phenyl-tropolone backbone of the molecule with four methoxy groups and an acetamido group attached to the periphery. Both the nitrogen atom of the acetamido group, and an overall size expressed as the calculated molar refractivity (CMR) of less than 9.7 were found to be essential structural parameters defining an MDR analog (Tang-Wai et at., 1993). A second common characteristic of MDR cell lines is that appearance of resistance is linked to a sustained decrease in the intracellular accumulation of drugs. This decrease is independent of a membrane electrical potential, but is strictly dependent on the presence of intact cellular ATP pools (Ling and Thompson, 1974; Di Marco, 1978; Fojo et al., 1985a). This decreased accumulation is linked to a concomitant increase in an ATP and temperature-dependent cellular drug efflux phenomenon, suggesting that an ATP-dependent transport mechanism is at work in MDR cells (Dano, 1973; Skovsgaard, 1978). Thirdly, very high levels of resistance observed in cell lines selected for long periods of time in high drug concentrations are often unstable and rapidly disappear upon culture of the cells in drug-free medium. These high levels of resistance are frequently associated with karyotypic alterations associated with gene amplification such as the appearance of small double minute chromosomes (Howell et al., 1984; Tsuruo et al., 1986), large chromosomal homogeneously staining (Grund et al., 1983, Meyers et al., 1985) or aberrantly banding regions (Teeter et al., 1986; Slovak et al., 1986), suggesting that .overexpression of a protein or group of proteins from amplified gene copies is required for high levels of resistance. Indeed, many early efforts were directed towards the identification of polypeptides whose expression was modulated in independently-derived MDR cell lines and several were found. Variations in the expression of a small calcium-binding protein (Koch et al., 1986), enzymes of the glycosylation pathway (Peterson et al., 1979), growth factor receptors (Meyers et al., 1988), membrane glycoproteins (Peterson et al., 1983; Richert et al., 1985) and several others were reported. However, the most ubiquitous marker of MDR was found to be the expression of a high molecular weight membrane protein that was reported initially by Ling et al. (Juliano and Ling, 1976) and designated P-glycoprotein, from its proposed role in modulating cellular permeability to drugs. P-glycoproteins (P-gp) are a heterogeneous group of membrane phosphoglycoproteins of molecular weight 160,000-210,000, first detected in a colchicine-resistant Chinese hamster ovary cell line (C5; Beth-Hansen et al., 1976), which have since been observed in virtually all MDR cell lines, irrespective of tissue or species origin (Beck and Danks, 1991;

P-Glycoprotein-Mediated

Multidrug Resistance

7

Sugimoto and Tsuruo, 1991). Subsequently, the development of specific polyclonal and monoclorral anti-P-gp antibodies has enabled the demonstration that P-gp can bind ATP (Comwell er al., 1987a; Schurr et al., 1989) and drug analogs (Cornwell et al., 1986a; Safa et al., 1986, 1989, 1993) and has ATPase activity (Hamada et al., 1988). These findings suggest that this protein may function as a broad specificity drug-efflux pump to reduce intracellular drug accumulation in MDR cells. This proposition, although intuitively appealing, has proven very difficult to demonstrate experimentally and the mechanism of action of P-gp remains a matter of considerable controversy. A final and clinically most important aspect of the MDR phenotype is that it can be reversed in P-gp-positive cells by a large group of structurally heterogeneous molecules that include calcium-channel blockers (verapamil, Tsuruo et al., 1981), calmodulin inhibitors (trifluope:razin, Tsuruo et al., 1982), immunomodulators (cyclosporin A, FK506, Slater et al., 1986), quinidines (Tsuruo et al., 1984)) reserpines (Pearce et al., 1989)) piperazine analogs (Kajiji et al., 1994) and many others (reviewed by Beck and Danks, 1991). When present in drug-containing culture medium, these compounds cause a dramatic increase in intracellular drug accumulation and a concomitant increase in cytotoxicity in MDR cells. Although the mechanism of action of these so-called modulators or reversal agents remains poorly understood, they all appear to compete for ‘drug-binding sites’ on P-gp (Cornwell et al., 1987b; Safa et al., 1987; Akiyama et al., 1988; Safa, 1988; Kamiwatari et al., 1989; Pearce et al., 1989; Tamai and Safa, 1990). While some of these compounds themselves appear to be transported by P-gp (verapamil; Yusa and Tsuruo, 1989) others are clearly not (progesterone; Ueda et al., 1992). Nevertheless, there has been an intense effort to generate novel and more efficacious MDR reversal agents, and several of them have shown promise in vivo and have been or are currently being tested in clinical trials. This important aspect of MDR and P-gp will be reviewed in Chapter 3 of this review.

P-Glycoproteins and mcfr Genes Some of the unique phenotypic features of P-gp-positive MDR cell lines demonstrating a very hi.gh degree of resistance and protein expression have been used to isolate the genes that encode P-gps. In one experimental approach by V. Ling and colleagues, a monoclonal antibody against hamster P-gp was produced (C219), which was then used to screen expression bacteriophage cDNA libraries constructed from a highly multidrug-resistant hamster cell (Kartner et al., 1985; Riordan et al., 1985). A second approach, used by our group and that of M. M. Gottesman (Fojo et al., 1985b) relied on a technique (in-gel renaturation; Roninson, 1983) allowing the detection of large ge:nomic DNA fragments commonly amplified in independently-derived highly drug-resistant hamster and human cell lines (Roninson et al., 1984, 1986). A 120 kb fragment of such a genomic domain was cloned from hamster LZ and C5 cells and found tal contain a transcription unit, which coded for a mRNA of approximately 5kb in size a.nd the level of expression was proportional to the degree of drug resistance in P-gp-positive cells (Gros et al., 1986a). DNA probes from this domain were used subsequently to isolate corresponding full length cDNA clones, which were found to encode :P-gp (Gros et al., 1986b; Chen et al., 1986). A third approach to cloning was based on the identification of cellular mRNA transcripts overexpressed in drug-resistant cells by differential cDNA hybridization. Five overexpressed mRNAs were identified and werie found to map on the same genomic fragment, with one sub-group of two genes (P-gps genes) consistently amplified and overexpressed in independent MDR cell lines

C. Shustik et al.

8

(De Bruijn et al., 1986; Van der Bliek et al., 1986). An important result from these cloning experiments revealed that P-gp was not a single protein, but rather forms part of a small gene family with three members in rodents (m&l, mdr2, mdr3) and two members in human (MDRI, MDR2). Nucleotide and predicted amino-acid sequence analysis of the prototype P-gp revealed some immediate and very interesting features (Chen et al., 1986; Gros et al., 1986b, 1988; Van der Bliek et al., 1988; Ng et al., 1989; Devault and Gros, 1990; Hsu et al., 1990; Devine et al., 1991). It is composed of approximately 1276 amino acids, which can be grouped in two halves, each encoding a large highly hydrophobic region (250 residues) and a large hydrophilic region (300 residues). Hydropathy profiling indicate that each hydrophobic half can be further divided into six highly hydrophobic segments characteristic of membrane-associated (TM) domains, suggesting that this protein is an integral membrane protein. On the other hand, each one of the hydrophilic regions contain a consensus sequence motif for ATP binding; this motif described originally by Walker et al. (1982) is formed by two consensus sequences, one highly conserved associated with interaction of the protein with the gamma phosphate of ATP (for possible hydrolysis), and an hydrophobic pocket believed to home the adenine moiety of ATP and possibly conferring the nucleotide specificity to the binding pocket (i.e. ATP vs GTP). The presence of these sites suggest that P-gp may be capable of binding and hydrolyzing ATP. Another readily identifiable feature of P-gp is the presence of a cluster of predicted N-linked glycosylation signals in the segment separating predicted TM1 and TM2, suggesting that it may be glycosylated. Therefore, the analysis of the predicted amino-acid sequence of P-gp predicts several structural features and are in agreement with its previous analysis by crude biochemical means. Accepting the proposition that P-gp is an integral membrane protein with an even (12) number of predicted TM domains, the combined positioning of: (1) the two predicted ATP-binding sites on the intracellular face of the membrane and (2) the predicted N-linked glycosylation sites on the extracellular side of the membrane, together yield the model proposed for P-gp, which is shown in Fig. 1. This structural

Fig.

1. The

putative

structure

of P-glycoprotein. nucleotide-binding

(NBI: nucleotide-binding site 2).

site

1; NB2:

P-Glycoprotein-Mediated

Multidrug

Resistance

9

model has gained general acceptance and has withstood the test of time with respect to the proposed position of the glycosylated loop and proposed ATP-binding sites. These predictions have been further corroborated by a number of epitope mapping studies using specific antibodies directed against synthetic P-gp peptides of known sequence or against fusion proteins containing P-gp subfragments (Bruggemann et al., 1989; Yoshimura et al., 1989). However, the precise number and orientation of the membrane domains remains a subject of much debate. Several studies, attempting to monitor membrane insertion and polarity of truncated P-gps (translated from in vitro-transcribed mRNA templates) in in vitro, have yielded contradictory results (Zhang and Ling, 1991, 1993; Zhang et al., 1993; Skach ef al., 1993; Skach, 1994). A recent and elegant study by Bibi and Beja (1994) of P-gp fusions to an indicator alkaline phosphatase enzyme expressed in E. coli has produced a more comprehensive picture of the membrane organization of P-gp, which resembles the original scheme predicted from hydropathy profiling (Gros et al., 1986b). Alignment of the two symmetrical halves of P-gp reveal that they are 38% identical and 62% homologous, suggesting that they arose at least in part from the duplication of a common ancestor. The region of highest homology is in the predicted ATP-binding sites, is lower within the predicted TM domains and disappears at the extreme amino terminus of each P-gp half (Devault and Gros, 1990). A similar pattern of sequence conservation is also detected amongst members of the P-gp family within one species. For example, the three mouse mdr genes map on the same segment of approximately 500 kb o:n chromosome 5 and have arisen by two successive gene duplication events, with the most recent one producing mdrl and mdr3 from a common ancestor (Raymond et al., 1989). The situation is somewhat different in humans, where only two genes (MDRI and MDR2) exist on the same genomic fragment on chromosome 7, presumably originating from a single gene duplication event (Chin et al., 1989; Ng et al., 1989). The three mouse proteins share amongst them between 73 and 83% sequence identity for a total homology varying between 85 and 92% (Devault and Gros, 1990). The regions of highest sequence homology between the three proteins are the predicted ATP-binding domains (over 95% homology), followed by the membrane-associated regions (70%) and finally the amino terminus and linker regions (20%). Interestingly, hybridization studies with gene-specific probes that could distinguish between mdrl, mdr2 and mdr3 mRNAs indicated that the emergence of multidrug resistance in independently-derived cell lines was associated with the overexpression of either mdrl or mdr3 from either single gene copies or from amplified copies of the mdr locus. However, none of the cell lines analyzed displayed mdr2 overexpression raising the possibility that mdr2 may not code for drug resistance (Raymond et al., 1989). The formal demonstration that P-gp is indeed responsible for the emergence of MDR and the dissection of structure-function relationships within this protein, awaited transfection studies. In these experiments, full length cDNA corresponding to either human or mouse mdr genes were introduced in plasmid or retroviral vectors containing viral promoter/enhancer elements that could direct high level expression of cDNA molecules inserted downstream (Gros et al., 1986~; Ueda et al., 1987; Guild et al., 1988). The introduction by transfection of such DNA constructs in drug-sensitive host cells, followed by plating the cells in drug-containing medium, resulted in the appearance of drug-resistant colonies, demonstrating a causal relationship between mdr

10

C. Shustik et al.

genes, P-gps and multidrug resistance. Moreover, transfection studies also revealed that P-gps encoded by distinct mdr genes were functionally distinct: although human MDRl and mouse m&-l and mdr3 could confer MDR in this transfection assay (Gros er al., 1986~; Ueda et al., 1987; Guild et al., 1988; Devault and Gros, 1990), neither human MDR2 (Schinkel et al., 1991) nor mouse mdr2 could do so (Buschman and Gros, 1992, 1994). In addition, careful comparison of the drug-resistance profiles encoded by mouse mdrl, mdr3 and human MDRl revealed both quantitative and qualitative differences between them, suggesting functional differences amongst these proteins with respect to substrate specificity (Dhir et al., 1992). Finally, a comparison of the biochemical and pharmacological characteristics of multidrug-resistant transfected cell clones stably overexpressing heterologous P-gps indicated that they were identical to those of P-gp-positive MDR cells, including the production of a phosphoglycoprotein of 160-200 kDa in size, which was capable of binding ATP and drug analogs and. which caused reduced ATP-dependent drug accumulation and increased ATP-dependent drug efflux (Schurr et al., 1989; Hammond et al., 1989). Taken together, these data clearly established that P-gp can cause MDR in vitro in cultured cells. Recent studies in mice have demonstrated that P-gp encoded by the mdr3 gene can also act on MDR drugs in vivo (Schinkel et al., 1994). One of the major sites of P-gp expression in normal tissues is the endothelial cells of the blood-brain barrier (Cordon-Cardo et al., 1989; Thiebaut et al., 1989) and in the mouse, the P-gp present at that site is encoded by the mdr3 gene (Arceci et al., 1988; Croop et al., 1989; Teeter et al., 1990). Through the advent of novel molecular techniques, which allow homologous recombination at defined loci in mouse pluripotent embryonal stem cells, it has become possible to perform selective gene disruption. This allows the creation of defined mouse mutants lacking a specific gene of interest (Capecchi, 1989) and such a mouse mdr3 null mutant has been created recently (Schinkel et al., 1994). Although these mice are viable and phenotypically normal, they displayed a lOO-fold increased susceptibility to the centrally neurotoxic peptide, ivermectin, and to the MDR drug, vinblastine (three-fold). In addition, the mutant mice reveal profound alterations in the pharmacokinetics and tissue distribution of vinblastine infused intravenously (1 mg/kg, 4 hr after i.v. infusion), the most pronounced being a 22-fold increase in brain vinblastine concentration, together with a reduced rate of elimination (Schinkel et al., 1994). These findings indicate that P-gp can act on MDR drugs in viva, in particular within the endothelial cells of the blood-brain barrier. In addition, these findings may also explain the apparent lack of response of certain brain tumors to chemotherapy and provide an explanation for some of the side-effects observed in patients treated with combinations of cytotoxic drugs and P-gp inhibitors (see Chapter 3).

The ABC Superfamily

of Membrane

Transport

Proteins

P-gp belongs to a large group of sequence-related proteins that share common structural and functional properties. This superfamily of proteins has been named the ABC (ATP Binding Cassette) family of membrane traffic ATPases. This family contains a large number of members and only a general overview of this family will be presented here; for a more thorough discussion of this aspect of P-gp, the reader is referred to an excellent recent review (Higgins, 1992).

P-Glycoprotein-Mediated

Multidrug

Resistance

11

The predicted nucleotide-binding domains of P-gp share a remarkable ancestral origin with a group of bacterial proteins known as the ‘binding protein-dependent periplasmic transporters’ or periplasmic permeases. These transporters are responsible for a large proportion of the high affinity nutrient import in Gram-negative bacteria such as E. coli and ,Salmonella typhimurium (Higgins et al., 1986). Periplasmic permeases are multi-subunit transporters composed of a soluble receptor expressed at high level in the periplasmic space as well as a membrane-associated complex typically composed of two highly hydrophobic membrane anchors and two peripheral or associated ATP-binding units. A Ilarge number of these transporters have been identified and transport in an ATP-dependent fashion such varied substrates as sugars (MalK, Gilson et al., 1982), amino acids (HisP, Higgins et al., 1982), short peptides (OppD, Ames et al., 1986), ions (PstB, Surin et al., 1985) and several others (Higgins, 1992). P-gps share a more significant homology to another group of bacterial transporters that include the ChvA gene of Agrobacterium tumefaciens (Cangelosi et al., 1989) and the HlyB gene of E. coli (Felmlee et al., 1985), which are involved in the secretion/export of p-1,2 glycan and the hemolytic enzyme hemolysin A, respectively. Sequence homology is particularly strong (35% identity) with HlyB and overlaps a region that includes the nucleotide-binding sites, but also the transmembrane region (Gros et al., 1988). HlyB is structurally similar to one half of P-gp (1 NB site, 6 TM domains) and shows a similar hydropathy profile. HlyB forms a membrane-associated complex with HlyD, which mediates the export of the 107 k.Da hemolysin A enzyme through the inner and outer membrane of E. coli. P-gp homologs have also been identified in lower and higher eukaryotes. These proteins share approximately 60% overall homology with P-gp, but display near identical hydropathy profiles resulting in very similar predicted secondary structure. These include the pfmdrl gene of, Plasmodium falciparum (Foote et al., 1989) in which mutations have been associated with the onset of chloroquine resistance in the malarial parasite (Foote et al., 1990). Members of this family have also been described in other eukaryotic parasites such as Entamoeba histolytica (ehpgp, emetine resistance; Samuelson et al., 1990), Leishmania tarantole (ltpgp, arsenate resistance; Ouellette et al., 1990), Leishmania donovani (ldmdrl, multidrug resistance; Henderson et al,, 1992) and others. Of particular interest is the yeast homolog of P-gp, STE-6, which is responsible for the transmembrane transport of the farnesylated dodecapeptide mating pheromone a factor (McGrath and Varshavsky, 1989; Kuchler et al., 1989). The structural homology between P-gp and STE-6 also translates into functional homology as the mouse mdr3 gene was shown capable of partially complementing a null allele at the STE-6 locus, restoring mating in a sterile ste6 mutant (Raymond et al., 1992). This finding is important, since it allows the biochemical and genetic dissection of P-gp function in this lower eukaryote. mdr-like genes of yet unknown function have also been identified in the fly (Drosophila melunogaster, Mdr49 and Mdr6.5) (Wu et al., 1991), worm (Caenorhabditis elegans, pgp-I-4) (Lincke et al., 1992) and plant (Arabidopsis thuliana) (Dudler et al., 1992). In humans, the ABC superfamily includes other transporters that may be associated with clinical pathology. Cystic fibrosis, the most common autosomal recessive genetic disease in the Caucasian population (one in 2500 live births), is caused by alterations in Cl- transport across the membrane of epithelial cells and is linked to mutations in the Cystic Fibrosis Transmembrane Conductance Regulator or CFTR (Riordan et

12

C. Shustik et al.

1989). CFTR is a member of the ABC family and functions as a cyclic AMP responsive outwardly rectifying chloride channel (Rich et al., 1990). Over 230 different mutations have been identified in the CFTR gene and are associated with mild or severe forms of CF; although these mutations span the entire CFTR molecule, there are two clusters of mutations in the two predicted NB sites of the protein (for review, see Tsui, 1992). The deletion of a single phenylalanine residue at position 508 accounts for nearly 70% of all cases of CF; this mutation was demonstrated to be a temperature-sensitive mutation resulting in failure of CFTR to mature and be properly targeted to the plasma membrane (Cheng et al., 1991; Denning et al., 1992a,b). The biochemical analysis of other naturally occurring mutations in CFTR indicate that mutations in the membrane region have a milder phenotype than those in the nucleotide-binding sites, and are sometimes associated with altered ion selectivity of the channel (Sheppard et al., 1993). Two additional members of the ABC superfamily have been identified in humans. These genes, designated TAP-l and TAP-2, have been mapped within the class II region of the major histocompatibility complex (MHC). These genes encode two proteins half the size of P-gp (1 NB site, 6 TM domains) and deletion or altered expression of these genes in T-lymphocytes abrogate their capacity to present antigens in association with class I MHC molecules. The TAP-l and TAP-2 proteins function as a heterodimer unit to actively transport antigenic peptides across the membrane of the reticuloendothelium for association with class I molecules and surface antigen presentation (Kelly et al., 1992; Spies et al., 1992; Powis et al., 1992). Finally, two genes designated PMP70 and ALD (Kamijo et al., 1990) encode P-gp homologs expressed in the peroxisomal membrane and have been proposed to transport coenzyme A-modified fatty acids. Interestingly, mutations at the ALD locus appear to cause X-linked adrenoleukodystrophy or its milder form, adrenomyelopathy (Mosser et al., 1993). al.,

Interaction of P-Glycoprotein with Drug Molecules The identification of protein domains in general and single amino-acid residues in particular implicated in recognition, binding and transport of drug molecules by P-gp is a necessary prerequisite for the rational design of novel types of cytotoxic drugs or modulators capable of by-passing or blocking the activity of P-gp in drugresistant tumors. The availability of photoactivatable analogs of MDR drugs and P-gp modulators, but also of cloned mdr cDNAs for mutagenesis studies and transfection systems to express and study the characteristics of mutant proteins have allowed the preliminary identification of P-gp domains likely to interact with drug molecules. This has been achieved by classical biochemical studies of photolabelled P-gp peptides and by the genetic analyses of P-gp mutants demonstrating altered substrate specificity. Derivatives of drug molecules and P-gp modulators containing highly reactive sidechains (e.g. nitrene groups) have been synthesized (reviewed by Safa, 1993). Upon incubation with a target protein, such reactive groups can be activated by high energy light, resulting in the formation of covalent bonds with neighboring amino acids; the inclusion of a radioactive tracer in these photoactivatable analogs allows the subsequent identification of peptides or discrete amino acids potentially forming or lining a drug-binding site. Technically, this is accomplished by incubating membranes from P-gp expressing cells with the photoactive ligand followed by photolabelling and immunoprecipitation of the protein and digestions with various proteases. The

P-Glycoprotein-Mediated

Multidrug

Resistance

13

labeled proteolytic fragments can then be localized within P-gp by immunological methods using antibodies directed against discrete peptides of the protein. Although many different photoactivatable derivatives of MDR drugs and modulators have been synthesize,d and shown to bind to P-gp, the most frequently used ones have been the drug analog [3H]-NABV (vindesine/vinblastine) (Cornwell et al., 1986; Safa et al., 1986), anld the P-gp modulators [sH]-azidopine (Safa et al., 1987; Yang et al., 1988) a calcium-channel blocker, and [1251]-iodoarylazidoprazosin, an cwl-adrenergic receptor ligand (Greenberger et al., 1990; Safa et al., 1990a). Elegant studies in intact cells by Raviv et al. (1990) suggested that drug molecules present in the proximity of P-gp were located within the membrane-lipid bilayer. 5-[1*5I]iodonaphtalene-1-azide (INA) is a membrane-specific probe that readily labels P-gp upon excitation with ultraviolet, but not visiblle light. Conversely, the fluorescent drug, daunomycin, emits UV light when excited with visible light, but this emission is weak. When daunomycin and INA are incubated with intact P-gp-positive cells and the cells irradiated with visible light, P-gp can be lalbeled by INA suggesting that daunomycin was close to P-gp, i.e. was located within the plasma membrane. These findings together with the high degree of lipid solubility common to all MDR drugs suggest that the predicted membrane-associated domains of P-gp may be the primary sites of drug-protein interaction. Independent studies established that at least two regions of the membrane-associated portion of P-gp were involved in binding photoactivatable ligands: one minor site located within the amino terminal half and a major site mapping to the carboxy terminal half (Bruggeman et al., 1989, 1992; Yoshimura et al., 1989; Greenberger et al., 1991). Further studies by Greenberger using a series of antibodies directed against discrete P-gp peptides demonstrated that prazosin and azidopine bound to the same site on P-gp, which was limited mostly to two 5 and 4-kDa tryptic peptides mapping immediately downstream the last membrane domains of each half of P-gp (Greenberger, 1993). These studies suggested that symmetrical regions in each half of P-gp may be involved in drug binding and transport (Greenberger, 1993). The important role of the predicted membrane-associated domains from both halves of P-gp in drug recognition and binding was established independently by the analysis of P-gp mutants revealing altered substrate specificity. In addition, the detailed analysis of some of these mutants allowed some predictions of the mechanism of drug binding to the protein. Sequence analysis of human P-gp expressed in a KB carcinoma multidrqg-resistant clone selected for high levels colchicine resistance revealed the substitution of a Gly to Val residue at position 185 (in the TM2 to TM3 interval). This substitution was found to cause opposite effects on the transport of colchicine, which was increased, and that of vinblastine, which was decreased (Choi et al., 1988). Labeling experiments using photoactivatable analogs of the two drugs demonstrate decreased colchicine binding and increased vinblastine binding to the mutant when compared to the wild-type protein, suggesting that a decreased rate of drug dissociation from the Valiss mutant was responsible for reduced drug transport (Safa et al., 1990b). A two-step mechanism of drug transport was proposed by these authors based on two distinct binding sites: one involved in initial drug binding (‘on’ rate) and another involved in drug release (‘off’ rate). In a separate study of hamster cells selected for high levels of resistance to actinomycin D, it was found that the increasingly high levels of resistance in P-gp-positive cells were not associated with further increase in P-gp expression, but rather with a Gly 33s--Ala339to Ala33aPro339 replacement within

14

C. Shustik

et al.

TM6 of the hamster P-gp encoded by the pgpl gene (Devine er al., 1992). On the other hand, we have recently demonstrated that a single Ser-Phe substitution within TM 11 (position 941, mdrl; position 939, mdr3) strongly modulates the overall activity and the substrate specificity of P-gps encoded by mouse mdrl and mdr3 (Gros et al., 1991; Dhir et al., 1993): While this Ser-Phe replacement had little effect on vinblastine resistance (two- to three-fold decrease in resistance), it strongly modulated the degree of colchicine and adriamycin resistance conferred by mutant P-gps (lO-30-fold reductions) on both mdrl and mdr3 backgrounds. In mdrl, the substitution to Phe at that position produced a mutant that retained vinblastine and actinomycin D resistance, but had lost adriamycin and colchicine resistance. In this case, it appeared that reduced resistance to colchicine and adriamycin was associated with reduced drug binding to the protein, suggesting that the ‘on rate’ of drug binding was affected by mutations in TM11 (Kajiji el al., 1993). It also appeared that mutations at that site affected the capacity of known P-gp modulators to reverse vinblastine resistance in individual mutants, suggesting that this site was important for P-gp interaction with both drug molecules and inhibitors (Kajiji et al., 1994). Finally, the role of predicted TM domains in drug interactions was tested recently in a more systematic way by Clarke and co-workers in two series of mutants. The 13 proline residues present within predicted TM domains of human P-gp were individually mutated to alanine (Loo and Clarke, 1993a). Mutations at two sites (Pro**s, TM4 and Pros@, TMlO) affected the substrate specificity of P-gp. The same strategy was applied to the 31 phenylalanine residues mapping in the predicted TM domains of P-gp, and mutations at two phenylalanine residues were found to modulate the activity of P-gp (Phe33*, TM6 and Phe978, TM12) towards distinct drugs (Loo and Clarke, 1993b). Surprisingly, the two key Phe residues identified in these studies mapped at the exact homologous position in each half of P-gp. Therefore, genetic analyses of mutant P-gps with altered profiles of drug resistance and biochemical studies of photolabeled P-gp peptides indicate that membrane-associated regions of the protein are important for drug binding and that this binding involves domains from the two symmetrical halves of the protein. The notion that both P-gp halves come together to form an active transport site is supported by the recent demonstration that the amino and carboxy terminal halves of P-gp expressed separately can get together in the membrane and create a transport site (Loo and Clarke, 1994). It also appears that drug binding to the protein involves an ‘on’ and ‘off’ rate of binding, which can be segregated in individual mutants. Interaction

of P-Glycoprotein

with ATP and ATPase Activity

Early studies established that the reduced drug uptake and increased drug release characteristic of P-gp-positive multidrug-resistant cells were strictly ATP dependent. This activity was insensitive to ouabain, an inhibitor of the Na+,K+-ATPase responsible for the generation of membrane potential in mammalian cells, could not be sustained by non-hydrolyzable ATP analogs and could be blocked either by poisons of mitochondrial respiration such as sodium azide and rotenone. These characteristics were verified subsequently in multidrug-resistant cell clones stably expressing transfected human or mouse mdr genes, together suggesting that P-gp or proteins associated with it bind ATP and have ATPase activity. Predicted amino-acid sequence of P-gp from cloned DNA identified two consensus sites for ATP binding, lending support to the notion that P-gp may itself bind and hydrolyze ATP. This predicted binding site, originally described by J. E. Walker in a series of ATP-binding proteins and ATPases, is

P-Glycoprotein-Mediated

Multidrug Resistance

1.5

composed of two consensus sequence motifs (Walker et al., 1982). The A motif of sequence G-X-X-G-X-G-K-T(S) is believed to form a flexible loop controlling access to the nucleotide-binding site, with the lysine residue interacting with phosphate groups of ATP and playing a key role in ATP hydrolysis. The B motif of sequence is believed to form a hydrophobic pocket homing the R/K-(X)%G(X)3+(Hyd)4-(D) adenine moiety of ATP and thereby conferring nucleotide specificity (ATP vs GTP) to the binding site. This motif has since then been described in many proteins, and its association with subunits of 6 TM domains such as in P-gp has been used to define a superfamily of ABC membrane ATPases, which include members from prokaryotes to eukaryotes (Higgins, 1992). Experimental analysis of P-gp-positive MDR cells or mdr transfectants indeed verified that P-gp could bind the photoactivatable analog of ATP, [s*P]-S-azido ATP (Cornwell et al., 1987a; Schurr et al., 1989). The demonstration and characterization of ATPase activity associated with P-gp has been more laborious and has been the object of intense investigation (reviewed ‘by Shapiro and Ling, 1994). The group of Tsuruo first measured ATPase activity in P-gp simply purified by immunoprecipitation with an anti-P-gp antibody (Hamada and Tsuruo, 1988). Since then, several other groups have demonstrated ATPase activity in either partially purified or highly purified P-gp preparations consisting of intact P-gp or P-gp fusion proteins. However, the biochemical characteristics of this ATPase activity measured by independent groups vary dramatically (Hamada and Tsuruo, 1988; Shimabuku et al., 1992; Ambudkar et al., 1992; Sarkadi et al., 1992; Al-Shawi and Senior, 1993; Sharom ,et al., 1993). It appears to have a specific activity varying between 30 and 1000 nmoYmin/mg protein with a K, for ATP between 0.3 and 1.5 pM and it is sensitive to vanadate and N-ethyl-maleimide. One of the interesting features of P-gp ATPase activity detected in four independent studies, is that it appears to be stimulated at a low, but reproducible level by certain MDR drugs and P-gp inhibitors. The role of the predicted ATP-binding sites in P-gp function has also been analyzed by a genetic approach using site-directed mutagenesis of the corresponding cDNA. First, it was established in a series of chimeric proteins where homologous regions of mdrl and mdr2 had been exchanged, that the two predicted ATP-binding sites of P-gp could be vertically exchanged between different members of the P-gp family, with the two NB sites of mdr2 complementing the activity of mdrl (Buschman and Gros, 1991). This suggested that NB sites play a major role in the common mechanism of action of the different P-gps. On the other hand, it was demonstrated that mutations of highly conserved lysine and glycine residues of the Walker A motif of either NB site of P-gp completely abrogate the capacity of P-gp to confer MDR (Azzaria et al., 1989). Interestingly, the mutants in the Walker A motif, although inactive, appeared to bind ATP with the same affinity as the wild-type protein, suggesting that a step subsequent to ATP binding, possibly ATP hydrolysis was impaired in these mutants. These results indicate that both ATP-binding sites are absolutely essential for P-gp function, and suggest that each P-gp half does not possess half the activity of the full protein, but rather that both halves cooperate to mediate function. The notion of cooperation between the two halves of P-gp to mediate function was strengthened recently by the observation that although neither half of P-gp expressed alone has ATPase activity, the simultaneous expression of both halves results in ATPase activity with characteristics similar to those of the wild-type protein (Loo and Clarke, 1994). Together these results demonstrate that the ATP-binding and ATPase properties are essential to P-gp function and that both predicted ATP-binding sites play a pivotal role in this activity.

16

Mechanism

C. Shustik et

of Drug Transport

al.

by P-gp

The early studies of P-gp-positive MDR cells clearly demonstrated that the emergence of MDR in these cells was linked to a marked decrease in the intracellular accumulation of the various drugs to which the cells expressed resistance to. This reduced cellular drug accumulation was strictly energy (ATP) dependent and was concomitant with an ATP-dependent increase in drug release from these cells. Two major hypotheses for the mechanism of P-gp action to explain this reduced drug accumulation have been put forward (reviewed by Endicott and Ling, 1989; Gottesman and Pastan, 1993). The first hypothesis is that P-gp functions as a drug transporter (efflux pump), which can act on a broad range of structurally unrelated molecules and uses the energy of ATP hydrolysis to mediate drug efflux. Experimental data supporting the notion that P-gp functions as a drug transporter include the observations that: (1) P-gp binds ATP analogs, has ATPase activity and mutations in either of its predicted ATP-binding domains abrogate its function; (2) P-gp binds drug analogs and mutations in its predicted TM domains modulate substrate specificity by altering drug binding to the protein; (3) P-gp shares homology with a number of prokaryotic and eukaryotic membrane proteins implicated in the ATP-dependent transport of various types of substrates across the membrane; and (4) transport studies in intact cells and plasma membrane vesicles from P-gp expressing cells indeed suggest that P-gp mediates increased ATP-dependent drug binding and/or transport into these vesicles (Cornwell et al., 1986b; Horio et al., 1988; Kamimoto et al., 1989). However, the formal demonstration that P-gp functions as a drug transporter has awaited the purification to homogeneity of the protein and reconstitution of transport in reconstituted proteoliposomes. A demonstration of active P-gp-mediated drug transport against a substrate concentration gradient in such a system would constitute a formal proof. Although several groups have succeeded in either partial or near complete purification of P-gp (Sharom et al., 1993; Shapiro and Ling, 1994), drug transport has so far only been accomplished from partially purified fractions (Sharom et al., 1993). A second opposing hypothesis proposes that P-gp itself is not a drug transporter, but rather has an indirect role in modifying the intracellular environment to create either an electrochemical or a pH gradient. In turn, these gradients would act in an indirect fashion to drive the movement of charged MDR drugs across the membrane. The major appeal of this hypothesis is its explanation of the unusual ability of P-gp to act on a vast number of structurally unrelated substrates, which yet share a high degree of hydrophobicity and are positively charged at neutral pH (lipophylic cations). Recently, P-gp expressing cells were found to release ATP to the extracellular medium in proportion to the level of P-gp expressed in these cells. This channel-like activity can be blocked by an anti-P-gp antibody and abrogated by mutations in the nucleotide-binding sites of P-gp previously shown to impair drug resistance (Abraham et al., 1993). In this model, P-gp was proposed to function as an outwardly directed ATP channel, creating an electrochemical ATP gradient and possibly providing a driving force for drug efflux. Another model for P-gp mechanism was proposed by Roepe et al. (1992, 1993), which is based on the observation that P-gp-positive cells demonstrate an altered intracellular pH (Thiebaut et al., 1990). In this model, P-gp would function either directly or indirectly to increase intracellular pH and/or lower

P-Glycoprotein-Mediated

Multidrug Resistance

17

electrical membrane potential, in consequence of which: (1) charged hydrophobic compounds such as MDR drugs (lipophylic cations) might be differently retained in P-gp-positive and negative cells, and (2) the pH-dependent binding of drug molecules to their .respective, but structurally unrelated targets might also be altered by P-gp overexpression. Each of these alternative models for the mechanism of P-gp action are attractive, but are difficult to reconcile with the genetic (mutants) and biochemical data (photolabeling) supporting a direct drug transporter model and has generated a considerable controversy in the field. This controversy has been extended by the recent proposal that P-gp may have a dual function that includes chloride ions transport (see Distribution and Possible Function of P-gp in Normal Tissues). Recent data obtained with P-gp expressed in heterologous systems have helped distinguish between a mechanism of action involving direct drug transport and the more indirect one, involving modulation of membrane electrical potential and/or pH modulation. A novel yeast expression system was developed recently for the expression and analysis of heterologous membrane proteins (Ruetz et al., 1993). This system takes advantage of the temperature-sensitive mutant see 6-4 (Walworth and Novick, 1987; Nakamoto et al., 1991), which is defective in the final step of the vesicular secretion pathway (fusion with the plasma membrane) and accumulates at the non-permissive temperature, large numbers of unfused vesicles that contain in their membrane, newly synthesized plasma membrane proteins, including exogenous ones introduced as cloned genes on plasmids (Nakamoto et al., 1991). These secretory vesicles (SV) present several major advantages over classical plasma membrane vesicles prepared by standard procedures: (1) they are highly homogeneous and can be easily purified in large amsounts; (2) they are homogeneous in size, tightly sealed and are fully functional organelles since the endogenous proton ATPase present in their membrane maintains a strong proton gradient; and (3) they are uniform with respect to orientation and P-gp molecules inserted in them, would expose their catalytic ATP-binding domains to the outside. P-gps encoded by the three mouse mdr genes, mdrl, mdr2 and mdr3, were individually expressed in these SVs and the kinetics of drug accumulation in these vesicles were measured under different conditions (Ruetz et al., 1994a). Under normal conditions, expression of mdrl or mdr3, but not mdr2, resulted in the appearance of a rapid drug accumulation in these vesicles, which could be completely abrogated by the P-gp inhibitor, verapamil. The enhanced P-gp-mediated drug accumulation was found to be independent of an intact proton gradient, which could be dissipated either by treatment with specific protonophores or by mutations in the gene encoding the endogenlous yeast proton ATPase (Nakamoto et al., 1991). Likewise, P-gp-mediated uptake of a positively-charged lipophilic cation was shown to occur against a cation (proton) gradient. These data argue strongly against the hypothesis that drug transport by P-gp occurs via a proton symport or antiport type of mechanism. Finally, colchicine transport by mdr3 in this system occurred against a continuously rising intravesicular drug concentration gradient, in favor of a direct drug transporter function for P-gp. Distribution

and Possible Function

of P-gp in Normal Tissues

The large body of data on the functional role of P-gp in multidrug resistance, together with the identification of its cellular and subcellular sites of expression in normal tissues, have led to several hypotheses regarding its function in normal tissues. Although they

18

C. Shustik et al.

appear very distinct, these proposed functions may prove not to be mutually exclusive. The first one is modeled on the role of P-gp in causing MDR and favors a detoxifying role of this protein for toxins normally present in the environment. The second is that MDR drugs are actually fraudulent substrates for P-gp and that the protein acts on yet to be defined normal cellular products. This is an important aspect of P-gp biology, and again, evidence for and against each of these models has been obtained. In addition, understanding the physiological role of P-gp is more than a strictly academic question, as inhibitors of P-gp function used to modulate drug resistance in P-gp-positive tumors in vivo will undoubtedly have deleterious effects on the normal physiological events controlled by P-gp. Expression of the various P-gp isoforms and their cellular mRNAs has been demonstrated to be tightly regulated in an organ- and cell-specific fashion. The highest levels of human MDRl protein and mRNA expression are found in the adrenal gland, kidney, jejunum, colon and endothelial cells of the blood-brain barrier, whereas human MDR2 is strongly expressed in liver. In normal mouse tissues, mdrl is expressed in the pregnant uterus, placenta, adrenal glands and kidney, while mdr2 is expressed primarily in liver, but also in muscle and mdr3 is found in lung, intestine and in brain (Fojo et al., 1987; Chin et al., 1989; Thiebaut et al., 1987, 1989; Cordon-Cardo et al., 1989; Croop et al., 1989; Arceci et al., 1988). Immunohistochemical analysis with specific anti-P-gp antibodies reveal that P-gps are expressed in a polarized manner on the apical membrane of secretory epithelial cells lining luminal spaces such as the glandular epithelial cells of the endometrium in the pregnant uterus, the biliary canaliculi of hepatocytes, the brush border of renal proximal tubules, pancreatic ductules, columnar epithelium of the intestine and in endothelial cells of the blood-brain barrier and in the testes (Thiebaut et al., 1987, 1989; Cordon-Cardo et al., 1989; Bradley et al., 1990; Georges et al., 1990, Buschman et al., 1992). Expression of P-gp at the surface of epithelial cells lining the luminal space of the intestine, kidney and liver suggests that P-gp may play a protective role at these sites. Additional experimental evidence in support of this proposal include: (1) the altered drug distribution and pharmacokinetics displayed by mutant mice bearing a null allele at mdr3 (see Characteristics of the Multidrug Resistance Phenotype in vitro); (2) endothelial cells from the blood-brain barrier can carry out unidirectional drug transport in vitro (Tatsuta et al., 1992); (3) the cardiac glycoside, digoxin, which is eliminated by glomerular filtration and tubular excretion is a P-gp substrate (Tanigawara et al., 1992); and (4) pluripotent stem cells of the hemopoietic system can transport the P-gp substrate fluorescent dye Rhodamine 123 (Chaudhary and Roninson, 1991). On the other hand, expression of P-gp at other sites such as the pancreatic ductules, the endometrial glands of the pregnant uterus and the adrenal cortex together with the observation that neither mouse or human MDR2 can confer drug resistance in transfection experiments, suggest that P-gp may act on different types of substrates at those sites. It has been proposed that in the uterus and the adrenal gland, P-gp may play a role in hormone transport; recent findings that P-gp can transport steroid hormones such as cortisol, aldosterone, dexamethasone (Ueda et al., 1992) and corticosterone (Wolf and Horwitz, 1992) would tend to support this notion. Moreover, since some of the MDR drugs recognized by P-gp have fairly large peptide backbones (actinomycin D, valinomycin, gramicidins), together with the observation that P-gp can transport small peptides (Sharma et al., 1992), including the yeast mating pheromone ‘a‘ (Raymond et al., 1992) raise the possibility that it may act on small or large physiological peptides.

P-Glycoprotein-Mediated

Multidrug Resistance

19

The structural homology detected between P-gp and the CFTR chloride channel (see The ABC Superfamily of Membrane Transport Proteins) suggested the possibility that P-gp may also have ion conducting properties. Valverde et al., 1992 observed that expression of the MDRl gene in NIH-3T3 cells resulted in the appearance of small chloride currents that could be inhibited by P-gp modulators, verapamil and forskoline. This P-gp-associated chloride conductance was found to be regulated by cell volume. Interestingly, the Cl- channel activity of P-gp was unaffected by mutations in the nucleotide-binding sites known to abolish drug transport (Gill et al., 1992). These authors proposed that P-gp may be a bi-functional protein capable of acting on both drug mo.lecules and ions in normal tissues and drug-resistant cells. A recent study demonstrated a remarkable pattern of complementarity between the expression profiles of CFTR and P-gp in normal epithelia (Trezise et al., 1992). This complementarity was particula:rly striking in the ileum, where P-gp was expressed in the epithelial cells of the villus, whereas CFTR expression was restricted to the crypt epithelia. A similar complementarity was noticed in the luminal and glandular epithelia of the uterus during pregnancy. Function

of P-gp in Liver

As can b’e.observed from data reviewed in the previous section, the normal physiological role of P-gp has been difficult to establish with any degree of certainty and has remained puzzling. Interestingly, it is the study of the enigmatic mdr2 class of P-gp (which does not confer multidrug resistance) that recently provided the most provocative findings to date, and has given yet another twist to the search for a normal function of P-gp. Immunological

studies with isoform-specific antibodies have demonstrated that expression is largely limited to liver, where it is found only at the apical pole (canalicular membrane) and not the sinusoidal side of epithelial cells lining the lumen of the bile canaliculi and biliary ductules (see Distribution and Possible Function of P-gp in Normal Tissues). It has been proposed that P-gp may be involved at that site in the transmembrane transport of a bile constituent (Buschman et al., 1992). Smit et al. (1993) have used homologous recombination in embryonal stem cells to create a mouse bearing a null mutation (mdr2-I-) at the mdr2 locus. Histological examination of this mutant revealed a severe liver pathology that included hepatocyte damage, strong inflammatory response, and more strikingly, destruction of the bile canaliculi. The most obvious biochemical consequence of the mutation was found to be at the level of lipid content of the bile; while heterozygous mice for the mutation (mdr2-+‘-) revealed a 50% decrease in the amount of phosphatidylcholine (PC) in the bile, PC was completely absent from the bile of animals homozygous for the mutation (mdr2-I-). This observation led the authors to propose that mdr2 may participate in the translocation of PC across the canalicular membrane into the bile. Th.is possibility was tested directly in yeast secretory vesicles stably expressing mouse mdr2: for this, a fluorescent lipid tag was inserted via liposome fusion into the membra.ne of these vesicles and the asymmetric distribution of the fluorescent lipid tag was monitored over time in the presence or absence of mdr2 (Ruetz et al., 1994b). Expression of mdr2 in secretory vesicles caused a time- and temperature-dependent enhancement of PC translocation from the outer leaflet to the inner leaflet of the membrane of these vesicles. The mdr2-mediated effect was specific since expression mdr2/MDR2

20

C. Shustik et al.

of mdr3 in these vesicles was without effect on the membrane distribution of PC. The increased mdr2-mediated PC translocation was strictly ATP and Mg2+ dependent, was abrogated by the ATPase inhibitor, vanadate, and the P-gp modulator, verapamil, but was insensitive to the presence of excess MDR drugs, colchicine and vinblastine. These experiments demonstrated that mdr2 is a lipid transporter and that it functions as a lipid flippase or translocase to move lipids from one leaflet of the membrane to the other (Ruetz and Gras, 1994). What are the physiological consequences of this mdr2 activity on our understanding of normal liver physiology? Bile acids are small hydrophobic molecules that play a key role in the emulsification of dietary fats; they are secreted by hepatocytes and delivered by specific transporters to the bile (Boyer et af., 1992), where they form mixed micelles with cholesterol and PC, thereby protecting the underlying epithelium of the canaliculi from the detergent action of bile acids, a protection that is missing in mutant mice lacking mdr2 (Smit et al., 1993). The mechanism by which PC molecules cross the membrane and are delivered to the bile is unclear and several models have been proposed (Coleman, 1987; Berr et al., 1993; Higgins, 1994). The finding that mdr2 can translocate lipids within the cell membrane suggests a mechanism by which mdr2 could create an asymmetric distribution of PC in the outer leaflet of the canalicular membrane, and that the simple detergent effect of bile acids could result in preferential extraction of PC from the canalicular membrane to form mixed micelles in the lumen of the bile ductules. Another important question that emerges from these findings is whether the flippase mechanism observed for mdr2 in the transport of PC may also apply to drug transport by the two other members of the family mdrl and mdr3. The high degree of sequence homology between the three proteins (approximately 90%) certainly argues for a common mechanism of action. This notion of sequence/function conservation is also supported by the observation that mouse mdr3 can complement the biological activity of its yeast homolog STE-6 (Raymond et al., 1992), although the two proteins share only 50% homology. Therefore, it is possible that mdrl and mdr3 may also be flippases for yet to be identified lipids and that drug molecules inserted in the membrane may be recognized as fraudulent lipid substrates. Of particular interest is the case of the P-gp substrate, gramicidin D. Gramicidin D is a linear peptide that forms a homodimer spanning the entire length of the lipid bilayer and kills cells by forming a transmembrane ion channel. It is easy to envision how a flippase-translocase activity for P-gp could abolish the formation or disrupt such a channel thereby causing cellular resistance to this drug without the necessity of ‘transmembrane transport’. It is interesting to note that cellular levels of drug resistance conveyed by either mdrl or mdr3 are highest for gramicidin amongst all drug tested in transfectants (Kajiji et al. ) 1993).

Chapter2

Multidrug Resistance

in the Clinic

The experimental evidence reviewed in the previous sections clearly demonstrates that in cultured cells, the emergence of multidrug resistance is caused by the overexpression of P-gps
Detection of MD/W/P-gp in Clinical Specimens Although overexpression of MDRl has unequivocally been demonstrated to cause drug resistance to natural product agents in vitro, the relevance of the MDRl gene to drug resistance in the clinical situation is more controversial. In part, this controversy stems from the divergent results, which have been reported for the detection of MDRl/P-gp in certain malignancies. Numerous methods to detect MDRlIP-gp in clinical specimens have been published (Dalton and Grogan, 1991). These methods may be categorized on the basis of whether MDRl mRNA or its protein product, P-gp, is detected and whether the assay requires bulk tissue or if single cells can be analyzed (Table 1). Ideally, it is preferable to detect P-gp in single cells. This type of assay allows for the determination of tissue heterogeneity and the ability to discriminate between normal and malignant cells. If normal cells expressing P-gp are present in the tumor specimen (such as cytotoxic lymphocytes), then the ability to distinguish normal from malignant cells is obviously important (Klimecki et al., 1994). Assays that best meet the requirements of detecting MDRlIP-gp at a single cell level are RNA in situ 21

C. Shustik et al.

22

Table 1. Methods to detect MDRIIP-gp MDRI

RNA

Bulk tissue

Northern Blot Slot Blot RNAse Protection Assay RT-PCR

Single cells

in situ

hybridization

in

clinical specimens P-glycoprotein

Western Blot

immunohistochemical staining FACS Analysis

hybridization, immunocytochemistry and flow cytometric analysis (FACS). At this time, only immunocytochemistry and FACS are capable of detecting P-gp on single cells. Both assays use monoclonal antibodies and therefore are subject to potential shortcomings, namely the cross reactivity of the antibodies with proteins other than P-gp (Thiebaut et al., 1989; Rao et al., 1994). Multiple antibodies that recognize unique epitopes may be necessary to prevent false positives due to cross reactivity with other proteins. The choice of which assay to use is likely to depend on the type and amount of tumor being studied. For solid tumors, immunohistochemical assays may be superior. Detection of P-gp by immunohistochemistry preserves the architecture of the tumor and individual tumor cells can be distinguished from normal stroma and macrophages. Weinstein and colleagues have reported that P-gp expression in colon cancer is predominant at the invasive front and may relate to the invasiveness of the tumor cells (Weinstein et al., 1991). Only studies using immunohistochemical methods, which preserve the normal architecture of the tumor tissue, are able to determine this type of differential expression. Results of immunohistochemical studies are likely to depend on a number of factors including type of tissue fixation and choice of monoclonal antibody. Ideally, immunohistochemical results should be confirmed by an assay to measure RNA such as RT-PCR or in situ hybridization (Noonan et al., 1990; Futscher et al., 1993). Presumably, any tissue that is positive for P-gp by immunohistochemistry would also be positive for MDRl RNA. For ‘liquid’ tumors, such as leukemias, FACS may be the best form of analysis. Monoclonal antibodies (MoAbs) that recognize an external epitope, such as MRK 16, UIC2 and 4E3, may be superior to those that recognize an internal epitope (C219, JSBl, C494) and therefore require cell fixation before analysis (Hamada and Tsuruo, 1986; Mechetner and Roninson, 1992; Arceci et al., 1993). An advantage of FACS analysis over other assays is the ability to study viable, unfixed cells. The study of unfixed, viable cells allows for the study of P-gp function by measuring the intracellular concentration of P-gp substrates such as the mitochondrial dye, Rhodamine 123 (Chaudhary and Roninson, 1991). Cells that are P-gp positive would be expected to be Rhodamine ‘dim’ compared to those that are P-gp negative and Rhodamine ‘bright’. In order to identify malignant cells, it is important to use markers of malignancy, such as DNA

P-Glycoprotein-Mediated

Multidrug

Resistance

23

ploidy or monoclonal antibodies that recognize tissue-specific antigens. If markers are not specific for malignant cells, then a risk for false-positive results will occur. A second assay to confirm FCA results such as RT-PCR would be advantageous. One of the major problems in determining the role of P-gp in clinical drug resistance is the diverse results obtained for a given tumor, even using the same assay, such as immunohistochemical detection (Grogan et al., 1990; Chan et al., 1994). For example, published results of the incidence of P-gp in breast cancer is quite variable, even though the primary method of detection in reported cases is by immunohistochemistry (Weinstein et al., 1993). Part of this variability may be due to the different monoclonal antibodies used, tissue preparation and fixation and third stage detection systems. In addition, criteria for positive cells are frequently different depending on the experience of the individual investigators. Given the variability in the actual methods used for immunohistochemical detection of P-gp in breast cancer, it is not surprising that there are discrepant results reported for this disease. General acceptance of laboratory criteria would appear to be critical for addressing the question of prognostic significance of P-gp in clinical tumors. It must be kept in mind, however, that methods of detection may vary for individual tumor types. In the past decade, a number of studies have reported on the detection of MDRlIP-gp in clinical tumor specimens. These initial studies used assays, which measured MDRIIP-gp in bulk t.issue such as immunoblots (Western blots) and RNA slot blots (Gerlach et al., 1987; Goldstein et al., 1989). These initial studies were important in that they demonstrated that MDRlIP-gp was present and could be detected in clinical tumor specimens. Despite the numerous potential problems of detecting MDRZIP-gp in clinical specimens, general agreement regarding the frequency and magnitude of expression of MDRlfP-gp has been established for certain malignancies. These malignancies can be categorized as having a high, moderate, or low level of P-gp expression. Tumors categorized as having a high level of MDRlIP-gp at time of diagnosis are derived from tissues that normally express high levels of P-gp, such as kidney, colon, liver and adrenal gland. These tumors are classified generally as being drug resistant from the time of diagnosis; however, mechanisms of drug resistance, in addition to P-gp, may be in place and it is not possible to ascribe clinical drug resistance to the presence of P-gp alone in these tumors. Demonstrating a relationship between P-gp expression and response to therapy will probably be more feasible in tumors that ‘acquire’ the MDR phenotype. Malignancies such as childhood sarcomas, breast cancer, malignant lymphomas, multiple myeloma and acule leukemias represent a class of tumors that are generally considered to be drug responsive, but have a significant percentage of patients who relapse presumably due to the dmevelopment of drug resistance. Analyzing for P-gp, before and after treatment with chemotherapy, in patients who acquire the MDR phenotype, will probably be more informative in determining the prognostic significance of P-gp than studying patients who have malignancies that are considered to be drug resistant de nova.

C. Shustik et al.

24

Incidence

of P-gp/MDRl

in Hematologic

Malignancies

Acute leukemia Progress in the therapy of acute leukemia has been marked by increases in the rate of remission induction, refined strategies for remission maintenance, and consolidation as well as improvements in supportive care measures. Long-term disease-free survival and cure of a majority of children with the diagnosis of acute lymphoblastic leukemia has been achieved. The same success has not been achieved for adult acute leukemias, but aggressive approaches to improve survival have been used. Despite the successful induction of remission in 60430% of adults with acute myeloid and lymphoid leukemia, relapse within 2 years from diagnosis is the norm, often with resistant disease and an inferior response to subsequent therapy. Interest in the multidrug resistance phenotype in acute leukemia is particularly strong because of the significant anti-leukemic activity of drugs which may be transported by P-gp. The anthracycline drugs, daunorubicin and idarubicin, are important components in the most commonly used induction regimens in acute myeloid leukemia and the Vinca alkaloid, vincristine, is a standard agent for remission induction in acute lymphoid leukemia. Other MDR-related drugs with significant anti-leukemic activity, including epipodophyllotoxins and mitoxantrone, have been incorporated into the treatment of acute leukemia. Since the presence of a critical level of functional P-gp in leukemic blasts is a potentially important determinant of response to treatment with these drugs, detection of P-gp expression in acute leukemia has been the object of numerous studies. Since the initial description of MDRl as a mechanism for tumor resistance to chemotherapy, several large prospective studies of MDRl expression in acute leukemia at diagnosis and in relapse have been reported. Levels of MDRlIP-gp activity determined by MDRl RNA are usually expressed in arbitrary units in reference to drug-resistant cell lines, which vary between studies and do not consistently correlate with results of immunocytochemical analysis of membrane or cytoplasmic expression of P-gp using monoclonal antibodies (MoAbs) directed to different epitopes. The sensitivity of mRNA detection by PCR requires relative purity of the malignant clonogenic population under study to ascertain significant clinical correlations. Heterogeneity in MDR level within leukemic cell populations and co-expression with other lineage maturation markers can best be studied by P-gp immunoreactivity using flow cytometry and by in situ hybridization for MDRl mRNA. The absence of standardized methodology for the assay of MDR activity and the lack of uniformity in the interpretation of results present problems for the comparative analysis of these studies. Disparate results as to the predictive value of MDR expression on response to treatment and with clinical outcome in acute leukemia have been reported, but the effect of treatment regimen variables and imbalances in other defined prognostic variables including age and cytogenetic abnormalities may have contributed. The correlation of treatment outcome with MDR-related drugs and in vitro MDR-mediated drug efflux and cytotoxicity in fresh leukemic cells by these drugs has been investigated to confirm the clinical relevance of this mechanism. The acquisition of MDR phenotype following chemotherapy exposure is suggested by an increased frequency of expression in secondary leukemia, but there has been, to date, a relative paucity of data in patients studied sequentially from diagnosis. Apart from the clinical relevance of such studies, acute leukemia has been a model for the study of MDR because of the availability of relatively pure, phenotypically characterized malignant

P-Glycoprotein-Mediated

cells permitting a more comprehensive other malignant tissues. Acute lymphoblastic

Multidrug

multi-parameter

Resistance

25

analysis than is possible with

leukemia

Acute lymphoblastic leukemia (ALL) is the most common malignancy of childhood and, with currently used regimens, remissions are achieved in 95% of cases with long-term, disease-free survival in 50-75%. In adults, ALL is diagnosed less frequently than myeloid ll:ukemia, but the prognosis is poorer than that of ALL in childhood despite similar treatment strategies. Most regimens employ vincristine and an anthracycline, daunomycin, as well as the epipodophyllotoxins, during induction and often in dalvage therapy following relapse. In an early study (Rothenberg et al., 1989), bone-marrow or peripheral-blood samples were evaluated in 28 pediatric and adult patients with ALL, including nine patients evaluated at initial presentation and 19 after relapse. MDRl mRNA levels were determined by an RNase protection assay and in individual cells by immunofluorescence with the MoAb, MRK-16 and by RNA in situ hybridization. In 4/28 samples (three after multiple relapses and one at initial presentation), heterogeneous expression of MDRl mRNA and P-gp was present, with very high levels in subpopulations of lymphoblasts comparable to those in the control multidrug-resistant cell line, KB 8-5. In two other studies using RNase protection assays, elevated MDRl mRNA levels were observed in 2/15 cases of untreated adult ALL (Goldstein et al., 1989) and low, but measurable levels in S/11 cases of adult ALL (Herweijer et al., 1990). By slot-blot hybridization, elevated ,UDRl mRNA levels were observed in 2/5 adult ALL cases (Marie et al., 1991). The intrinsic expression of P-gp by leukemic lymphoblasts at presentation was reported in a study of 36 pediatric and 23 adult cases of ALL (Goasguen et al., 1993). P-gp expression was demonstrated by an immunocytochemical APAAP technique using the MoAbs, .JSB-1 and C-219. Cases were classified as positive if >l% of cells stained with either antibody, with the majority of positive samples containing >lO% positive cells, but with two distinctive patterns of reactivity observed. Seventeen cases were positive for both .JSB-1 and C-219 and four with JSB-1 only. No correlation was noted between P-gp expression and lymphoblast immunophenotype (T vs B + pre-B), although 6/7 BALL were P-gp positive. In this series, differences between adult and pediatric cases of ALL were observed with respect to the likelihood of achieving first complete remission (CR) and lymphoblast P-gp expression. Among the adult patients, complete remission was achieved in 5/9 P-gp-positive cases compared to 13/14 of negative cases (56 vs 93%), whereas CR was attained in 92% of pediatric patients irrespective of P-gp status (11/12 P-gp positive; 22/24 P-gp negative). Relapse rate was, however, significantly higher in children who were initially P-gp positive (S/11,73% vs 7/22,32%). During the period of follow-up, relapse occurred in all of the P-gp-positive adults entering CR compared to 6/13 negative cases. For the entire group, overall survival was significantly correlated with MDR phenotype, with the latter characteristic shown by multivariate analysis to be independent of age. Since post-translational modification of P-gp has been observed and may alter pump activity, MDRl mRNA and P-gp expression in clinical samples has been correlated with functional activity. P-gp function has been studied in leukemic cells by measurement of efflux of the fluorescent dye Rhodamine 123 on flow cytometry and inhibition of this function by verapamil. In a study of acute leukemia samples using dual-

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fluorescence analysis with Rhodamine 123 and phycoerythrin-conjugated antibodies to lymphoid antigens and common ALL antigen (CDlO), significant Rhodamine 123 efflux could be demonstrated in 17/29 (59%) cases of AML at diagnosis, but in only l/19 (5%) of ALL patients (Ludescher et al., 1993a). However, in three samples without demonstrable Rhodamine 123 efflux, levels of MDRl mRNA by quantitative PCR were comparable to a sample with functional activity. MDR expression in leukemic cells of adult T-cell leukemia, associated with the human T-cell leukemia virus type I (HTLV-I), was studied by immunoblotting with C-219 for P-gp, and by slot-blot analysis for MDRl mRNA (Kuwazuru et al., 1990). In peripheral blood and lymph node samples from 25 patients with ATL, 8/20 (40%) samples at diagnosis were positive and all six, initially negative patients when studied at relapse or with refractory disease, had acquired an MDR-positive phenotype.

Acute non-lymphoblastic leukemia In newly diagnosed AML, the frequency of mdr expression reported in different studies has ranged widely from undetectable to present in the majority of cases. While discrepant results on the frequency of MDRIiP-gp expression in de novo untreated AML are apparent in published reports, differences may be, in part, related to variability in the the assay systems employed. Determination of MDRl mRNA levels by slot-blot hybridization depends on the use of different positive control cell lines and arbitrary units assigned. Quantitative analysis by PCR can detect low levels of MDRl gene expression not observed by slot-blot technique, but constitutive expression of MDRl by normal subpopulations of blood and bone marrow may confound these results if the samples analyzed contain these latter. Immunohistochemical techniques using flow cytometry reflect the variable levels of P-gp in a heterogeneous population, but results are not standardized and may underestimate clinically important levels. Studies using antibodies directed to different epitopes of P-gp with different cellular localization have yielded discordant results and levels of reactivity considered as positive results have not been consistent. Efflux studies .using fluorescent dyes and inhibition by MDR modulators have been correlated, but not consistently, with MDRl mRNA and P-gp levels, but correlation with an increased level of clinical resistance to P-gp-transported drugs is more difficult to establish. A number of studies support the conclusion that the MDR phenotype is more consistently observed in leukemia supervening on an antecedent myelodysplastic syndrome, secondary to cytotoxic chemotherapy for other tumors and following the transformation of chronic myelogenous leukemia. In the myelodysplastic syndromes, the frequency of transformation to acute leukemia is related to the FAB subtype of the former, refractory anemia with excess blasts (RAEB and RAEB-T) and chronic myelomonocytic leukemia (CMML) being at highest risk. These ‘secondary’ leukemias may be refractory to conventional anti-leukemic regimens with response rates consistently inferior to those achieved in de novo ANLL. Immunocytochemical expression of P-gp by leukemic cells was initially demonstrated in two patients with clinically drug-resistant ANLL (Ma et al., 1987). An increase in the intensity and proportion of cells staining with C-219 was observed during disease progression after clinical drug resistance was apparent. MDRl mRNA was

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27

quantitateld by slot-blot analysis in patients with myelodysplasia (MDS) and AML (Holmes et al., 1989). Increased mRNA levels were observed in 2/8 untreated de lzovo AML compared to 5/5 untreated cases of secondary AML (with antecedent MDS or postcytotoxic chemotherapy). Five of eight patients with refractory AML were found to have elevated levels. In patients with MDS, most of whom had received previous chemotherapy, 7/19 marrow samples demonstrated increased mRNA expression. MDRl gene amplification was not demonstrated in those samples with increased mRNA levels. However, in a study of 14 cases of AML evaluated at initial presentation and at relapse (Ito et al., 1989), P-gp was undetectable by flow cytometry using MRK-16 on leukemic cells both at diagnosis and at relapse despite previous therapy with cumulative doses of daunomycin ranging from 160 to 1800 mg. In a subset of these patients with sufficient RNA for study, MDRl mRNA was also unmeasurable. In a larger study of 74 patients with AML (61 analyzed at diagnosis and 13 at first and subsequent relapse) assayed by Northern blot analysis, no correlation was noted between MDRl transcript level and FAB mor]phologic subtype of AML (Sato et al., 1990). In concordance with the results of Holmes et al. (1989), higher levels of MDRl mRNA were evident in ‘poor-risk’ AML developing after an antecedent myelodysplastic syndrome. Of six patients assayed at diagnosis
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A large series analyzed 150 newly diagnosed cases of ANLL for P-gp expression by indirect immunofluorescence with MRK 16 on flow cytometry. Diagnosis was based on FAB criteria with concurrent immunophenotypic analysis of leukemic cells for expression of the myeloid and stem cell antigens, CD13, 14, 15, 33 and CD34 (Campos et al., 1992). Remission induction in all patients included daunomycin or mitoxantrone for 3 days and cytosine arabinoside for 7 days. Using 20% positively-staining cells as a cutoff, P-gp expression was found in 71/150 (47%) samples, with heterogeneity in fluorescence intensity and percentage of positive cells. No correlation of P-gp result and age, FAB subtype (except for M3, promyelocytic leukemia) and blast percentage was observed, but as previously reported, P-gp was more frequently observed in cases of ANLL following a myelodysplastic syndrome (9/12) or after chemotherapy (10116) than in de now ANLL (521122). There was a positive correlation between P-gp and expression of the stem cell antigen CD34. In this, as in the Pirker study, there was a significant difference in complete remission rate between P-gp-negative and P-gp-positive cases (64/79, 81% vs 23/71, 32%). The association of MDR with leukemic immunophenotype has been examined by other investigators. In an analysis of 75 newly diagnosed cases of adult acute leukemia, MDRl mRNA levels were determined by RT-PCR with confirmation by immunostaining for P-gp with the MoAb, UIC2 (Miwa et al., 1993). Functional assessment of P-gp was performed by study of Rhodamine 123 efflux in the presence of verapamil. The results revealed a good correlation between the different assays. By the FAB classification, Ml and M2 subtypes were most frequently associated with MDRl expression at diagnosis (6/10 and 7/18, respectively). None of five cases of M3 (acute promyelocytic leukemia) were positive, a finding consistent with other studies that have found infrequent P-gp expression in this leukemic subtype. Fifteen of the 50 cases (30%) of AML studied had elevated levels of MDRl mRNA. In AML samples, a close relationship between MDRl mRNA positivity and expression of CD34 was found, although this association was not apparent in ALL. For both AML and ALL, expression of the early differentiation antigen, CD7, was significantly associated with MDR phenotype and function. In a sequential analysis of P-gp expression at diagnosis during remission and at relapse, bone-marrow samples from 20 patients with ALL and 12 with AML were evaluated by immunocytochemical staining for C-219 using an alkaline phosphatase anti-alkaline phosphatase (APAAP) technique (Must0 et al., 1991). P-gp-positive cells were infrequent at diagnosis (4/20 ALL, 2/12 AML) with a wide range of per cent positive cells. Relapse was associated with P-gp expression ranging from 61 to 100% of leukemic cells in 6/8 AML samples studied, with failure of re-induction therapy in all cases. A functional analysis of MDRl was performed in conjunction with mRNA quantitation by slot-blot hybridization in leukemic cells from peripheral blood or bone marrow from 41 adults with acute leukemia (Marie et al., 1991). This study was based on a heterogeneous population, which included 23 patients with de now ANLL, five with ALL and 13 with ‘secondary’ leukemia following myelodysplasia or transformation of a myeloproliferative disorder. MDRZ levels, expressed in arbitrary units by reference to a control drug-resistant leukemic line, K562/R7, were increased in 8/19 untreated de nova ANLL and in 2/4 with prior therapy that included MDR-related drugs. In the entire series, high levels (>lO U) were found in 50% of patients previously treated

P-Glycoprotein-Mediated

Multidrug Resistance

29

with MDF:-related drugs compared with 19% without previous exposure to these drugs. Serial determinations in four patients with AML showed increasing transcript levels following treatment with a daunomycin-containing regimen. In 22 AML samples, in vitro resis’tance of leukemic clonogenic cells to cytotoxicity by different drugs was measured and a correlation with MDRl levels suggested. P-gp expression by immunocytochemistry was compared in marrow samples from 43 patients with myelodysplasia, primary and secondary to cytotoxic therapy and in acute leukemia following MDS (List et al., 1991). The MoAbs, C-219 and MRK-16, were used for immunoperoxidase staining in a modified biotin-avidin technique and >5% reactivity of immature mononuclear cells considered a positive result. Seven of 32 cases of primary MDS (22%) were positive, predominantly among cases of chronic myelomonocytic leukemia (CMML), compared with 4/5 cases of MDS complicating cytotoxic chemotherapy or radiotherapy. In AML preceded by MDS, 4/7 (57%) of samples were interpreted as positive. P-gp positivity was significantly associated with CD34 expression in this study. The association of MDRl and CD34 expression in myelodysplasia is supported in a subsequent study of 26 patients with untreated MDS of whom three had progressed to AML (Sonneveld et al., 1993). Patients were categorized as low risk and high risk for leukemic transformation based on FAB criteria, and marrow or blood samples were analyzed by immunocytochemistry for P-gp using a panel of MOAbs, C219, C494, JSBl and MRK 16 and by MDRl mRNA quantitation. Blasts were characterized for expression of myeloid antigens, CD13, CD15 and CD33 as well as the early non-lineage specific markers, CD34 and terminal deoxynucleotidyl transferase (TdT). P-gp staining of isolated blasts was found in 14/17 marrow samples from patients with high risk MDS compared to 2/9 with low risk MDS and was associated with an immature phenotype, i.e. CD34 or CD13/33 and TdT. In this study, P-gp expression was more frequently observed in association with an abnormal karyotype, commonly a loss or deletion on chromosome 7. MDRIIP-gp function in ANLL has been addressed in a study of 49 previously untreated patients (Ross et al., 1993). Leukemic blasts from marrow samples were analyzed for P-gp expression by Western immunoblotting using C 219 and studied for daunomycin accumulation, retention and cytotoxicity in the presence of the MDR-modulating agents, verapamil, cyclosporin A and progesterone. Using drug-resistant and sensitive cell lines for controls, P-gp was expressed by only 3138 samples tested, but enhancement of daunomycin accumulation and retention by cyclosporin A and verapamil was demonstrable in most samples as well as enhancement of in vitro cytotoxicity by daunomycin. However, the per cent increase observed in daunomycin accumulation on exposure to cyclosporin A or verapamil was relatively modest (2040%) and the correlation with P-gp expression uncertain. Lymphoma Studies of drug resistance in malignant lymphoma have extensively investigated the better understood mechanism of multidrug resistance mediated by P-gp. In an early study of lymphoma biopsy specimens, the expression of P-gp by tumor cells was correlated with in vitro resistance to doxorubicin (Salmon et al., 1989). Frozen sections and cytospin preparations of tumor cell suspensions were stained by a modified biotin-avidin conjugated immunoperoxidase method using two murine monoclonal

30

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antibodies against P-gp, JSB-1 and C219, and graded for positivity in relation to control doxorubicin-resistant myeloma cell lines. Drug sensitivity was determined by inhibition of proliferation of fresh tumor cells on exposure to doxorubicin in a tumor clonogenic assay. Of six patients with lymphoma, positive-staining reactions were observed in three, associated with in vitro doxorubicin resistance. Resistance to doxorubicin was present in one of three P-gp-negative tumors. In a subsequent report of P-gp expression and reversal of drug resistance in lymphoma, these authors reported positive tumour staining in l/42 (2%) newly diagnosed patients and 701 (64%) previously treated patients with clinical drug resistance (Miller et al., 1991). In an analysis of 23 lymphoma specimens with quantitation of MDRl mRNA levels by a slot-blot procedure (Goldstein et al., 1989), measurable levels were detectable in three of five tumors from treated patients and in four of 18 from untreated patients, but the levels, expressed in arbitrary units relative to a drug-reiistant cell line, were low in five of the seven positive tumors. The pathologic subtypes and the details of chemotherapy were, however, not specified. P-gp expression has also been examined in untreated non-Hodgkin’s lymphomas by immunohistochemical staining with the MoAbs MRK 16 and C 219 using an APAAP technique on frozen sections (Pileri et al., 1991). Heterogeneous expression of P-gp was observed in 25/60 cases (42%) with a wide range of positively-staining malignant cells. In this study, no correlation was observed between level of P-gp expression and histologic subtype using an updated Kiel classification. The frequency of P-gp expression in a group of untreated diffuse large cell and immunoblastic lymphomas has been reported (Niehans et al., 1992). Frozen biopsy specimens obtained at diagnosis from 57 patients were tested for immunoreactivity with the antibodies, MRK16 and C219, by an immunoperoxidase technique. Positive staining of >50% of tumor cells was observed in 13/57 (26%) with lower levels of staining (ll-50% of tumor cells and ~10%) in an additional 15 and 14 cases, respectively, and the intensity of staining appeared proportional to the level of positivity. In fifteen specimens, no detectable staining of cells was observed. In this study, P-gp expression did not correlate with response rate to combination chemotherapy, which was not standardized, and no differences in survival were observed between groups classified into high, intermediate and low P-gp expression. In a study of MDRl and glutathione-S-transferase gene expression, increased MDRZ mRNA levels, determined by Northern blot hybridization, were detected in 8/23 NHL biopsies, including four from patients that had received no chemotherapy (Moscow et al., 1989). In this study, there was no apparent relation between mRNA level and drug exposure and in the limited number of cases, there was a tendency to higher levels in low grade pathology. In another study reported from Taiwan, a retrospective analysis of P-gp expression was performed in 21 cases of recurrent NHL (Cheng et al., 1993). Frozen sections were stained by an avidin-biotin-peroxidase method for detection of immunoreactivity with C219 and with NCL-GST n directed to a glutathione-S-transferase. Diagnosis was based on the International Working Formulation and by immunophenotypic profile, 11 cases being identified as B-cell NHL and 10 as T-cell with predominantly intermediate and high grade pathologies represented. P-gp reactivity in >20% of cells was present in 3/11 recurrent B-NHL and in 4/10 T-NHL while staining of <20% of cells was observed in l/11 cases with pre-treatment biopsy material available for comparative study. In this study, those cases of peripheral T-cell lymphoma (PTCL) with clonal integration of EBV were particularly associated with MDR phenotype. Interesting results were reported recently in a study of tumor biopsies from patients with relapsed or refractory

P-Giycoprotein-Mediated

Multidrug

Resistance

31

lymphoma treated in a Phase I trial of R-verapamil as an MDR-modulating agent administetred with EPOCH (infusional etoposide, vincristine, doxorubicin, and bolus cyclophosphamide and oral prednisone). MDRl mRNA could be demonstrated by PCR in 23/27 biopsies before treatment with EPOCH, with a further increase in MDRl mRNA levels on re-biopsy in 5/8 lymphomas after treatment (Chabner et al., 1994). From these published studies, it is apparent that low levels of MDRl expression may be present in a fraction of tumor cells in patients with newly-diagnosed malignant lymphoma and that the distribution and level of expression increases after treatment failure. However, the frequency of MDRl expression in different NHL histologies and the relative importance of this mechanism in clinical drug resistance require further investigation.

Multiple myeloma The treatment of multiple myeloma is often initiated with the combination of an alkylating agent, melphalan, with a corticosteroid, usually prednisone or prednisolone. An objective response, usually defined as at least a ~50% decrease in pre-treatment M peak, is achieved in approximately 50% of patients with melphalan and prednisone. Regimens with multiple alkylating agents and including vincristine and doxorubicin, have resulted in higher response rates in selected studies, but overviews of randomized trials with large numbers of patients have not demonstrated overall improvement in survival ,with combination chemotherapy compared to melphalan-prednisone. Nevertheless, doxorubicin is an active drug against myeloma and is used in first-line combination chemotherapy (VCAP, VBAP, ABCM) and in the treatment of myeloma refractory or relapsing after alkylating agent therapy. The combination of doxorubicin and vincristine given by continuous infusion for four days with oral dexamethasone (VAD) has given responses in 50-75% of patients relapsing after treatment with melphalan-prednisone or with primary unresponsiveness to that regimen. The contribution of high dose dexamethasone to the efficacy of this regimen, relative to vincristine and doxorubicin, has been demonstrated by the responses observed with dexamethasone used alone. However, salvage therapy following progression after treatment with VAD has limit’ed impact on survival. Resistance to VAD in multiple myeloma mediated by of the multidrug resistance phenotype has been extensively investigated, often in conjunction with trials aimed at circumventing this resistance. Early studies with human myeloma cell lines confirmed that doxorubicin resistance, resulting from continuous in vitro exposure to gradually increasing doses of that drug, was associated with the increased expression of P-gp and with coll,ateral resistance to other MDR drugs, but with retained sensitivity to alkylating drugs. Analysis of myeloma lines with incremental levels of multidrug resistance showed a correlation between P-gp, measured semi-quantitatively by immunohistochemical staining, and the mean lethal dose of doxorubicin, and an inverse correlation with intracellular doxorubicin accumulation (Dalton et al., 1986). In these multidrug-resistant myeloma lines, the level of P-gp expression by individual cells is relatively uniform unlike the considerable heterogeneity that is observed between tumor cells in clinical specimens.

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There have been several interesting studies evaluating MDR in multiple myeloma by predominantly immunohistochemical methods. Using DNA aneuploidy as a marker of malignant cells, two-parameter flow cytometry was used to quantitate the percentage of P-gp-positive marrow plasma cells from 22 patients with multiple myeloma (Epstein et al., 1989). P-gp expression was detected by indirect immunofluorescence using the monoclonal antibody, C219. In this study of ten patients evaluated prior to treament with VAD and 12 patients following treatment, heterogeneity in the level of P-gp fluorescence intensity on tumor cells of individual patients was observed. An increased number of P-gp-positive cells was observed in three patients not previously exposed to MDR drugs, and in the group of patients treated with VAD, the proportion of positive cells, but not level of intensity, correlated inversely with objective response. However, the predictive value of P-gp expression for response to VAD was not corroborated in a study of patients refractory to alkylating agents (Cornelissen et al., 1994). Bone-marrow samples from 63 patients were analyzed for P-gp staining with the monoclonal antibody, C219, using an alkaline phosphatase technique. With a cutoff of ~30% P-gp-staining plasma cells for scoring as a positive sample, 37/63 (59%) of samples were positive, including 14/22 with primary refractory disease and 23/41 on relapse after prior response to alkylating agent therapy. Response to treatment with VAD was not significantly correlated with P-gp expression, with 17/37 responders in the P-gp-positive group compared to 15/26 in the P-gp-negative group. The discrepancy between these results of the predictive value of P-gp expression on treatment outcome are only partly explained by differences in the timing of marrow sampling in relation to treatment with VAD and different response criteria. The effect of previous treatment with drugs of the MDR group on P-gp expression by myeloma was evaluated in a study of 96 samples from patients with variable disease duration, stage and treatment (Grogan et al., 1993). Bone marrows were studied by an immunohistochemical assay for P-gp using the MDRl specific monoclonal antibody, JSB-1. Positive samples usually contained >30% plasma cells staining for P-gp (22/24), with negative samples defined by the absence of any positive cells. The incidence of P-gp positivity was significantly lower in patients with no prior therapy (3/47, 6%) compared to treated patients (21/49, 43%). Analysis of these data with regard to previous treatment demonstrated that the incidence of P-gp positivity was correlated with the cumulative doses of doxorubicin and vincristine in 40 patients who had received either one or both drugs. Of 31 patients treated with doxorubicin, 1508 (83%) administered >340 mg were positive compared to 3/13 (23%) exposed to <340 mg total dose. In this study, P-gp expression was not correlated with time from diagnosis, disease duration, or with plasma cell characteristics such as labeling index or aberrant phenotype. The relative absence of P-gp and weak expression of MDRl mRNA by plasma cells in bone marrow of untreated myeloma patients has been supported by most studies. P-gp expression was investigated at presentation in 53 patients by immunohistochemical and flow cytometric analysis using C-219 MoAb (Ucci et al., 1992). By immunohistochemistry, 22/53 (41%) had variable levels of P-gp staining on marrow plasma cells with a range of l-60% (median 6%). Using bivariate flow cytometry for DNA ploidy and C-219 immunofluorescence, P-gp levels were significantly higher in the hyperdiploid fraction in 5/10 patients with both diploid and hyperdiploid plasma cell populations. In this study, response rate was higher in the P-gp negative group (75 vs 25%, p < 0.01) regardless of the chemotherapy regimen

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used (melphalan-prednisone in 24 patients or peptichemio, vincristine and prednisone in 13 patients), although response duration and overall survival were not significantly different. The investigation of P-gp expression by plasma cells has been extended to the comparative study of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma. In the former, a serum monoclonal immunoglobulin peak is detected in the absence of clinical features of myeloma. The level of M peak may remain stable for years, but progression to symptomatic myeloma occurs in approximately 20% of cases on long-term follow-up. In one study, P-gp expression by plasma cells was determined by immunohistochemical staining with the MoAbs, C219 and JSBl, on marrow samples from 38 patients with MGUS and from 105 patients with untreated myeloma (Sonneveld et al., 1993). Positive P-gp staining was demonstrated on >50% of plasma cells in 32/38 MGUS samples compared to 33/105 (31%) of samples from patients ,with myeloma. No progression to overt myeloma was observed in the group of P-gp-positive MGUS cases during a two-year follow period, but progression to aggressive myeloma was observed in 3/6 cases with P-gp-negative plasma cells. With the previous demonstration of CD56 on plasma cells in myeloma, the authors also examined the ability of this antigen to distinguish myeloma from MGUS. Expression of the CD56 antigen in high density was found on plasma cells in 43/57 patients with active myeloma analyzed compared to O/23 of patients with MGUS. Although CD56 in this analysis correlated inversely with P-gp expression, this antigen is usually associated with NK cells, which have high levels of P-gp expression and function. The presence on myeloma cells of CD56 as well as other non-lymphoid antigens has been interpreted as reflecting the malignant transformation of an earlier hematopoietic progenitor in myeloma. Intrinsic expression by plasma cells of MDRl may represent a normal developmental marker and correlation with markers of earlier hematopoietic stages or other lineages may be i:nformative. A potentially relevant role of MDR in myeloma is raised by studies implicating a circulating B-lymphocyte population in the pathogenesis of myeloma. Several groups have presented evidence for an aneuploid B-cell population in the peripheral blood, with clonal Ig heavy chain gene rearrangement and restricted light chain expression similar to the plasma cells of the marrow. The hypothesis that these circulating B-cells represent a precursor ‘clonogenic’ compartment for the malignant, terminally differentiated, plasma cells of myeloma has implications for any curative therapy of this disease. An important study found that the majority of peripheral blood B-cells from patients with myeloma express P-gp detected by the monoclonal antibody, MRK-16 (Pilarski and Belch, 1994). The functional activity of the P-gp was confirme,d by flow cytometric analysis of Rhodamine 123-dye efflux, which could be inhibited by verapamil or cyclosporin A, and by resistance to in vitro doxorubicin cytotoxicity. In this study, P-gp expression was particularly associated with a subset of large CD19+ B-cells, defined by physical properties of forward and side scatter on flow cytometry, which express CDllb, a B2 integrin adhesion molecule required for extravasation. P-gp expression by blood monoclonal B-cells, but not by CD38+ plasma cells in bone marrow, was detectable in untreated patients with a dynamic pattern Iof variable, but persistent expression during the course of disease in response to treatment. It appears that P-gp may be expressed at certain stages of normal B-cell differentiation and the persistence of a clonogenic compartment of malignant B-cells in myeloma resistant to conventional doses of doxorubicin suggests that this population must be specifically targeted for therapy by alternative strategies.

C. Shustik et al.

34

Chronic lymphocytic

leukemia

Chronic lymphocytic leukemia (CLL) is predominantly a clonal B-cell lymphoproliferative disorder that has been characterized as a disease of ‘accumulation’ of long-lived lymphocytes. In the absence of curative therapy, treatment of CLL is often deferred until disease progression. Alkylating agents, primarily chlorambucil and cyclophosphamide, in combination with corticosteroids, have been used in the first-line treatment of CLL with objective response rates of 50-70%. In randomized clinical trials, doxorubicin and vincristine in combination with cyclophosphamide and prednisone (CVP, CHOP) have produced higher response rates in patients with more advanced disease compared to chlorambucil alone. More recently, the purine nucleoside analogues, fludarabine and 2-chlorodeoxyadenosine, have shown promising activity in low-grade lymphoproliferative disorders with complete response rates in CLL superior to those achieved with single alkylating agent or combination chemotherapy, and these will probably be incorporated earlier in the treatment of CLL. The multidrug resistance phenotype of lymphocytes in CLL has been reported in several studies with variable frequency of detectable expression depending on the assay system. Increased levels of MDRl mRNA were demonstrated by slot-blot analysis of RNA extracted from peripheral blood of 4/7 untreated patients with CLL and in 13/27 treated patients (Holmes et al., 1990). The probe used in this study did not distinguish MDRl and MDR2 mRNA. In a subsequent study of the same patient material (Cumber et al., 1990), P-gp expression by peripheral blood lymphocytes was determined by immunofluorescent staining with MRK-16. In contrast to the finding of increased MDRl mRNA in 53% of samples, P-gp was detectable on lymphocytes of only 12% of cases (4/34 of which 3/5 previously treated with vincristine and anthracycline). However, neuraminidase treatment of lymphocytes resulted in an increased frequency of lymphocyte P-gp expression (13/25) suggesting a masking of the P-gp epitope recognized by the MoAb MRK-16, possibly by post-translational sialation. Using the MoAbs, MRK-16 and C-219, for flow cytometric analysis and immunohistochemical staining, P-gp expression was evaluated in 63 cases of B-CLL (Michieli et al., 1991). By immunohistochemistry, all samples contained a variable proportion of lymphocytes (range, 20-95%) staining with MRK-16, but with weak reactivity in 44/63. By flow cytometry, the percentage of positive cells was lower, but ranged between 21 and 86% in 45 cases using MRK-16, with slightly lower frequency using C-219. These findings did not correlate with previous therapy, time from diagnosis or disease stage. and MDR3 mRNA levels were measured by an RNase protection assay in 17 patients with CLL (Herweijer et al., 1990). Quantitation of RNA levels was in arbitrary units standardized by reference to human liver RNA, which has a relatively high expression of both MDRl and MDR3. Elevated levels were detected in all samples with overlap in the range of values of the untreated (7) and treated (10) patients. In a subsequent report on 31 patients with CLL, these authors found expression of both MDRl and MDR3 in 29 patients with high levels of MDRl in nine (Sonneveld et al., 1992b). Although MDRl levels were not significantly associated with disease stage, MDR3 expression was correlated with more advanced stage. The level of MDRl mRNA was determined by Northern blotting analysis and after PCR amplification of MDRl MDRI

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complementary DNA in 48 patients with CLL at different stages of disease (Shustik et al., 1991). Four of twenty-nine untreated patients had intrinsica& high levels of MDRl mRNA. In the group of patients analyzed after treatment, 5119 had increased levels, but these results were unrelated to vincristine and doxorubicin exposure. In patients monitored serially, decreases in MDRl mRNA levels were observed to occur both in the absence of therapy or following treatment with chlorambucil. In a study attempting to correlate ~53 gene mutation with drug resistance (El Rouby et al., 1993), MDRl and MDR3 mRNA were quantitated by PCR in B-cells from 16 patients with CLL. Intermediate to high levels of MDRl gene expression were present in all samples analyzed, while 12/16 expressed MDR3 with at variable levels. In this study, there was no association observed between MDRl or MDR3 levels and disease stage or prior therapy. P-gp activity in CLL has been studied by dual fluorescence assaying efflux by CD19+ B-CLL cells of the fluorescent dye Rhodamine 123 in the presence of an MDR inhibitor (Ludescher et al., 1993). In 34142 (81%) samples studied, Rhodamine 123 efflux inhibited by 10 pM verapamil and/or 1 pM dexniguldipine was detected in lO-70% of cells with significantly lower percentages among previously untreated patients. Among treated patients, Rhodamine 123-efflux activity was unrelated to cumulative dose of vincristine or mitoxantrone. In 26 samples, MDRl and MDR3 mRNA bevels were quantitated by PCR with good correlation between MDRl mRNA levels and Rhodamine 123 efflux, although three samples without Rhodamine 123 efflux did have low, but detectable levels of MDRl mRNA. The MDR phenotype of B-hairy cell leukemia (B-HCL), a low-grade lymphoproliferative disorder of cells with distinctive morphology, but without an identifiable normal counterpart, has been studied by an RNase protection assay (Herweijer et al., 1991). Low to intermediate levels of MDRl transcript levels were found in 8/S peripheral blood or spleen cells from patients with B-HCL and even higher levels MDR3, but the significance of these results is difficult to interpret since the population of cells analyzed was not characterized. MDRl expression is detectable at low level in be linkeid to differentiation in these cells. The remains to be established by the determination CD19+ population corresponding to the CLL

Incidence of P-gp/MDRf

normal peripheral blood B-cells and may significance of elevated levels in B-CLL of MDRl expression in a normal CD5+, phenotype.

in Epithelial and Other Solid Tumors

The constitutive expression of P-gp in normal tissues of epithelial and neuroectodermal origin and a physiological role as indicated by localization studies in these tissues has been discussed above. MDRl gene expression by tumors arising from these sites has been determined by quantitative analysis of mRNA transcript levels using slot-blot and PCR assays (Noonan et al., 1990). Of normal tissues studied, the highest levels are found in adrenal gland and kidney, particularly in the medullary regions. Intermediate levels are observed in liver, lung and in the gastrointestinal tract epithelium of colon, jejunum and rectum.

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The MDR phenotype of malignant tumors arising from these sites has been extensively documented and has been proposed as a possible explanation for the intrinsic resistance of many of these tumors to chemotherapy. In the following section, we will review the results of these studies in tumors of non-hematopoietic origin. Neuroblastoma Neuroblastoma is a neuroendocrine malignancy of childhood with predominant sites of origin in the adrenal medulla and neural sympathetic ganglia in the abdomen, pelvis, mediastinum and neck. Normal adrenal medulla, which is the primary site of approximately 40% of neuroblastomas, has been demonstrated to express high levels of MDRl mRNA. This tumor is potentially curable depending on age at diagnosis and tumor stage. Disease characteristics including tumor histopathology, catecholamine production, N-myc oncogene activation and serum ferritin have also been correlated with prognosis in neuroblastoma. Localized disease is treated primarily by surgical resection with chemotherapy given for more advanced disease. Among the drugs with anti-tumor activity in neuroblastoma are those of the MDR group including doxorubicin, dactinomycin, vincristine, etoposide and teniposide, which are often used in combination with cyclophosphamide and c&platinum. Despite advances in treatment modalities, 5-year disease-free survival rate in children with distant metastatic sites (Stage IV) is less than 15%. Although complete responses may be achieved with initial chemotherapy, the unfavorable prognosis of non-localized disease is attributable to primary treatment failure and to recurrence with unresponsive disease in the majority of cases. The role of MDR in neuroblastoma as a contributory factor to treatment failure, and as a predictive variable for treatment, has been evaluated in several studies. Detectable levels of MDRl mRNA using an RNAse protection assay were found in 17/34 neuroblastoma specimens from ‘untreated patients and 16/16 from previously treated patients, with higher levels of expression in the latter group (Goldstein er al., 1989). Another study of neuroblastomas using an RNA slot-blot assay for tumor MDRl mRNA quantitation with reference to the same drug-resistant line KB-8-5 as the finding of increased in the earlier study (Bourhis et al., 1989a), corroborated levels following chemotherapy with regimens that contained vincristine, doxorubicin and etoposide (11/26 treated vs l/15 untreated). Augmentation of MDRl level was observed in a paired tumor specimen obtained before and after treatment with three cycles of vincristine and cyclophosphamide. In this series, tumors from patients with Stage IV disease were associated with higher MDRl mRNA levels than those at Stages I, II, III or IV-S. Similar results of MDRl mRNA expression were reported in another series of 49 tumors studied either at diagnosis or at the time of surgical resection following chemotherapy (Goldstein et al., 1990). These studies suggest that increased levels of MDRl expression in neuroblastoma may result from acquisition of the MDR phenotype by a malignant population following drug exposure or by the selective persistence of mdr-positive cells with de now resistance to chemotherapeutic drugs of the MDR group. The predictive value of P-gp expression for response to treatment was evaluated in a retrospective study of 67 children with neuroblastoma (Chan et al., 1991). Sequential tumor samples were assayed for P-gp by a multilayer immunoperoxidase method using two murine monoclonal antibodies, C219 and C494, and semiquantitatively graded by the highest level of staining observed in individual

P-Glycoprotein-Mediated

Multidrug

Resistance

37

tumor cells. Immunohistochemical results were correlated with disease stage, response to primary treatment and survival. In this study, P-gp was undetectable in pre-treatment samples from patients with Stage I (tumor localized to organ of origin) (O/2), and Stage II (node-negative or ipsilateral node-positive tumors beyond the organ of origin) (O/21), but was present on l/17 tumors in Stage III and 12/19 tumours in Stage IV. Positive samples contained at least 20% positive cells while negative tumors showed a complete absence of positively-staining cells. Treatment outcome in 44 patients with non-localized disease (III, IV and IV-S) was compared on the basis of tumor staining for P-gp. Complete response to primary treatment was attained in 26/31 patients (84%) with tumors negative for P-gp versus 6113 (46%) with P-gp-positive tumors. With stratification for tumor stage and patient age, relapse-free survival and overall survival were significantly better in the former group. Relapses following chemotherapy among the initially P-gp-negative group (6131) were associated in all cases with the finding of P-gp expression in the recurrent tumor. This important study demonstrated a significant correlation between P-gp expression in neuroblastoma and the failure of chemotherapy. However, the basis for an adverse effect of P-gp expression at diagnosis on response to treatment with regimens that include the highly active non-MDR drugs, cyclophoaphamide and c&platinum, is incompletely understood. Soft tiswe

sarcoma

Soft tissue sarcoma refers to a group of histologically diverse tumors observed infrequently in adults, but representing the fifth most common malignancy of childhood. Rhabdomyosarcoma and undifferentiated sarcoma are chemosensitive embryonal tumors of mesenchymal stem cell origin, which account for approximately 90% of pediatric sarcomas. Treatment protocols often include drugs of the MDR group including vincristine, etoposide and dactinomycin in combination with the alkylating agents, cyclophosphamide or ifosfamide. P-gp exp:ression in pediatric rhabdomyosarcoma and undifferentiated sarcoma has been reported in a retrospective study of 30 children treated at the Hospital for Sick Children in Toronto (Chan et al., 1990). Sixty-two biopsy samples obtained at diagnosis or following treatment were analyzed by the assay system described above in the study of neuroblastoma. Treatment results were compared between the nine patients with P-gp-positive tumors and the 21 patients with negative tumors. For the purpose of this retrospective analysis of survival, P-gp results of diagnostic and relapse samples were combined. Given the small numbers in the study, the authors were unable to stratify patients by prognostic variables and treatment regimen, but observed a significant difference in relapse-free survival favoring patients with P-gp-negative tumors. All nine patients with P-gp-positive tumors during the course of disease relapsed after initial response to therapy. Of these, five patients presented with P-gp-negative tumors at diagnosis, but were P-gp positive at relapse. Disease progression with clinical drug resistance was associated with increase in the frequency and intensity of P-gp staining by tumor cells in sequential biopsies. Other sarcomas of childhood have been less well-studied than rhabdomyosarcoma. Osteogenic sarcoma and chondrosarcoma are commonly treated with drugs of the MDR family, but less successfully than Ewing’s sarcoma, a tumor of late childhood and adolescence. By PCR quantitation of MDRl mRNA, measurable levels were found in 24/31 untreated osteogenic sarcomas and in

38

C. Shustik et al.

13/15 chondrosarcomas, but in O/6 untreated Ewing’s sarcoma (Noonan et al., 1990), while a low MDRZ transcript level was detected in l/2 Ewing’s sarcoma biopsies following treatment with vincristine and actinomycin D in a screening study of diverse tumors (Fojo et al., 1987a). Studies of P-gp in adult sarcomas are limited by the relative rarity of these tumors of mesenchymal origin with diverse histology. In one study, P-gp was detectable by immunoblotting with C219 in 6/2.5 sarcomas (Gerlach et al., 1987). Three of the positive specimens were from patients whose previous treatment had included adriamycin, vincristine and actinomycin D, while three were surgical specimens from patients not treated with chemotherapy. However, in a larger series, MDRl mRNA could not be demonstrated in any of 20 pathologically-unspecific sarcomas (Goldstein et al., 1989). Breast cancer Chemotherapeutic drugs are used both for the adjuvant treatment of high risk breast cancer following resection of the primary tumor and for the treatment of unresectable locally advanced or metastatic breast cancer. Doxorubicin has significant single agent activity in this disease as does the anthracycline derivative, epirubicin, which has a more favorable therapeutic index because of lesser cardiotoxicity and myelosuppression at equimolar doses. Doxorubicin has commonly been used in combination with alkylating drugs and anti-metabolites not transported by P-gp, but several other drugs of the MDR group are used in initial treatment or in relapsed disease, including vincristine, mitoxantrone and taxol. P-gp is expressed at low level in normal breast tissue and most studies in breast cancer have confirmed a low incidence of expression in biopsy samples from untreated patients. In the largest study reported of P-gp in breast cancer (Merkel et al., 1989), frozen biopsy specimens were examined by Southern blot for MDRl gene amplification, by Northern blot for MDRl mRNA transcripts and by Western blot for P-gp expression using the MoAb C219. In 219 biopsies from untreated patients and 29 from patients treated with doxorubicin, MDR gene amplification could not be demonstrated. MDRZ mRNA was undetectable by Northern blot hybridization in 53 untreated primary tumors and 11 metastatic tumors from patients treated with doxorubicin; P-gp immunoreactivity was not present in any of 125 primary tumors. The uniformly negative results of MDRl expression in this large series have not been corroborated by other studies and may be attributable to the relative insensitivity of the assay systems. A later study evaluating P-gp expression by Western immunoblotting using C219 in breast cancer (Sanfilippo et al., 1991) found detectable P-gp in lo/34 (29%) of untreated tumors and in 904 (64%) patients treated with various regimens. Analysis of 57 biopsies from untreated patients (Goldstein et al., 1989) by slot-blot quantitation for MDRl mRNA found low transcript levels in nine samples (15%), but moderate to high levels in 6/S samples following treatment with at least one MDR-related drug. A higher incidence of MDRI mRNA expression in untreated breast carcinoma (9/16) was demonstrated by in situ hybridization (Kacinski et al., 1989). An immunocytochemical study of cytospins of fresh tumor cell suspensions and of frozen sections using P-gp MoAbs JSB-1 and C 219 showed positive staining in 5113 cases of breast cancer (Salmon et al., 1989). In those cases with positive staining, four had received prior chemotherapy. P-gp staining was observed in l/4 primary tumors tested in mastectomy specimens from untreated patients. In a series of locally advanced, clinical Stage IIIA and IIIB breast cancer (Ro et al., 1990), P-gp expression was undetectable

P-Glycoprotein-Mediated

Multidrug

Resistance

39

by immunostaining with C219 in eight tumors prior to chemotherapy. However, in 20/40 specimens from patients who underwent mastectomy following treatment with three cycles of chemotherapy consisting of doxorubicin, cyclophosphamide, vincristine and prednisone, a variable percentage of P-gp-positive tumor cells (~5% - >30%) were observed, with the suggestion of a poorer response to neoadjuvant chemotherapy in those patients with a higher frequency of P-gp-positive cells. Similar results were obtained with the same antibody in a smaller study (Schneider et al., 1989) with positive staining of isolated tumor cells in 2/12 specimens from untreated patients. An increased frequency of positively-staining tumor cells was found in patients previously treated with MDR-related drugs. A higher frequency of detectable P-gp expression in untreated. breast cancer was reported using MoAbs C219 and MRK16 in an indirect alkaline phosphatase technique (Wishart et al., 1990). Heterogeneous expression of P-gp in a small percentage of tumor epithelial cells was detected in most biopsies with both MoAbs (21/29 with C219; 16/29 with MRK16). In a prospective study, biopsy specimens from 20 untreated patients with locally advanced and metastatic disease, were stuldied for P-gp expression prior to chemotherapy with a regimen including doxorubilcin and vincristine (Verrelle et al., 1991). Positive staining for C494, with an avidin-biotin-immunoperoxidase technique was observed in >75% of cells on frozen section of 15/20 biopsies. In this small study, P-gp expression was correlated with an adverse outcome in response to treatment and progression-free survival. Renal cell carcinoma P-gp expression in normal kidney can be demonstrated to localize to the apical surface of proximal tubular epithelial cells (Thiebaut et al., 1987), at which site a normal transport function may be served. Renal cell carcinoma usually arises from tubular epithelium and treatment of this neoplasm with chemotherapy is disappointing although occasiorral responses to vinblastine and doxorubicin have been reported. In an early study, elevated MDRZ mRNA levels quantitated by slot-blot analysis were reported in 6/8 renal cell carcinomas, but with a wide range of expression and with considerable overlap in levels measured in adjacent normal kidney tissue (Fojo ef al., 1987b). Similar results were obtained by the same technique in a study demonstrating elevated levels in 35/50 tumors comparable to those of the multidrug-resistant KB-8-5 cell line used for reference (Goldstein et al., 1989). In a study of 42 renal cell carcinoma specimens, MDRl mRNA levels in renal cell carcinoma have been correlated with tumor grade (Kanamaru et al., 1989) with higher levels expressed in more differentiated tumors. MDRl mRNA levels were measured in 25 urogenital tumors before chemotherapy (Kakehi et al., 1988). Twelve of 14 renal cell carcinomas, predominantly clear cell subtype, had elevated levels in the same range as the moderately multidrug-resistant KB cell line. By contrast, low levels were found in transrtional cell carcinomas arising in bladder, renal pelvis and ureter, which are characteristically sensitive to treatment with MDR-related drugs. Colorectal

carcinoma

Colorectal carcinoma is the second most frequent cause of mortality from cancer and treatment by chemotherapy is relatively ineffective. Response to MDR-related drugs is

40

C. Shustik et al.

minimal and other drugs, usually fluorouracil (5FU) in combination with leucovorin, have been used for adjuvant therapy and for metastatic disease with modest response rates. Intermediate levels of MDRI mRNA have been found in colon carcinomas as well as in normal colonic mucosal epithelium. P-gp is localized by immunohistochemical staining to the apical surface of superficial columnar epithelial cells. In a study of eight primary colon carcinomas (Fojo et al., 1987a), comparable levels of MDRl mRNA were observed in tumor and adjacent normal colonic mucosa and did not correlate with histology or degree of anaplasia in the tumor cells. In another study, measurable levels were reported in 35/40 tumor samples with high levels in ten (Goldstein et al., 1989). The variability of tumor sampling and localization of P-gp expression within a tumor is demonstrated by an immunohistochemical study of colon carcinoma (Weinstein et al., 1991). Paraffin-embedded surgical specimens from 95 patients with primary colon adenocarcinoma invading into the muscularis propria or beyond (Stage Bl or greater) were analyzed by an avidin-biotin-peroxidase technique using MoAbs JSB-1 and C219. P-gp immunostaining was detected in the major tumor mass in 65/95 specimens, but was typically heterogeneous with the most intense staining at the tumor-stroma interface and in deeply invasive carcinoma cells. Results of P-gp immunostaining were generally concordant for primary tumor and regional lymph node metastases, but in 3/6 involved nodes with P-gp-negative primary tumors, metastatic carcinoma cells were positive with C219. P-gp positivity in the population of solitary invading carcinoma cells at the leading edge of the tumor was associated with a locally aggressive pattern of vascular invasion and lymph node metastasis.

Ovarian carcinoma Ovarian carcinoma is a potentially curable neoplasm when localized and treated by surgical excision, but remains the leading cause of gynecological cancer mortality. In more advanced disease, debulking surgery and combination chemotherapy are employed. The tumor is generally sensitive to initial chemotherapy and current frontline platinum-based regimens are associated with response rates of 50-75%. Anthracyclines and epipodophyllotoxins have demonstrated activity in ovarian carcinoma and the notable activity of taxol, a potential P-gp substrate, in patients refractory to or relapsing after platinum-based therapy, has attracted interest in the MDR phenotype of this tumor. MDRl mRNA analyzed by slot-blot analysis was undetectable in 16 tumors from untreated patients (Goldstein et al., 1989), with similar results in 35 tumors studied by another group (Bourhis et al., 1989b). Using a two-step immunoperoxidase technique with MoAbs C219 and JSB-1, positive staining was observed in tumors from 2/33 untreated patients with ovarian cancer of different histologic types. In a larger group, which included 24 patients with tumor sampling after chemotherapy usually including one or more MDR-related drugs, only 4/57 tumors were considered positive (Rubin et al., 1990). By comparison, a low intensity of P-gp expression by ovarian cancers was found in 16/21 untreated patients using C219 in a three-step immunoperoxidase technique, which increases assay sensitivity by signal amplification (van der Zee et al., 1991).

P-Glycoprotein-Mediated

Multidrug Resistance

41

By PCR analysis, low levels of MDRl mRNA transcripts could be demonstrated in 21/26 tumors from untreated patients (Noonan et al., 1990). In another study with PCR quantitation of MDRl mRNA in 60 ovarian carcinomas, measurable transcript levels were found in 30/46 (65%) tumors from untreated patients (Holzmayer et al., 1992). Acquisition of the MDR phenotype or selection by chemotherapy in ovarian carcinoma. is suggested by the results of several studies comparing the frequency of MDRl expression in tumors from untreated patients and from patients treated with MDR-related drugs. Using slot-blot hybridization, MDRl mRNA was detectable in 3/10 tumors from patients previously treated with doxorubicin or vincristine, but no relationship was observed between mRNA levels and cumulative doxorubicin dose. No MDRl transcript signals were detectable in five tumors from patients treated with cyclophosphamide and k-platinum. In a study of ovarian adenocarcinoma cells in ascitic fluid from five previously treated patients (Bell et al., 1985), Western immunoblotting for P-gp yielded a positive result in 2/5 tumor samples and an increase in expression in tumor cells from one patient after disease progression on treatment with doxorubicin. By PCR analysis, MDRl mRNA could be demonstrated in lO/lO tumors from patients treated with at least one MDR drug, but there was no correlation between transcript levels in the positive samples and cumulative drug exposure (Holzmayer et al., 1992). An immunohistochemical study of P-gp using C219 on cryostat sections of tumor specimens (van der Zee et al., 1991) found tumor cell staining in 16/21 untreated. cases with a range of 5-50% of positive cells. In patients treated with a cyclophosphamide-platinum regimen, 8/13 tumors contained P-gp-positive tumor cells with1 a comparable range of expression as untreated tumors. Depending on the assay system used, the frequency of MDRl expression in untreated ovarian carcinoma varies widely, but with less sensitive methods, an increase in detectable expression is observed in tumors from patients previously treated with chemotherapy. Whether non-MDR drugs such as cyclophosphamide and platinum contribute to the elimination of P-gp-negative tumor cells and selection of multidrug-resistant cells or induce the MDR phenotype is difficult to ascertain from the available data.

Hepa tocellular carcinoma P-gp is detectable by immunohistochemistry on hepatocytes and bile duct epithelium with localization to the bile canalicular surface of hepatocytes and the luminal surface of biliary duct epithelium. Hepatocellular carcinoma and cholangiocarcinoma, which arise from these cells are difficult to treat with systemic chemotherapy, but intraarterial hepatic infusion with doxorubicin has resulted in tumor regression. P-gp expression has been implicated in the intrinsic resistance of these tumor types to cytotoxic: chemotherapy. In one study, MDRl mRNA by slot-blot quantitation was detectable by Northern blot in 12/12 hepatomas with high levels in seven (Goldstein et al., 1989). Immunohistochemical staining for JSB-1 by avidin-biotin-peroxidase was observed in 29/43 (67%) of tumors obtained at surgical resection or at autopsy with no significant difference between specimens from treated (17127) and untreated (12116) patients (Itsubo et al., 1994). However, in the latter study, cellular localization of immuno reactivity and incidence differed among histologic types of hepatocellular carcinoma characterized as trabecular, pseudoglandular and compact.

42

C. Shustik et al.

Lung cancer Response to chemotherapy in lung cancer is related to pathology with initial chemosensitivity in the majority of cases of small cell lung carcinoma (SCLC) to drugs of the MDR group and relative refractoriness of other types (NSCLC) to most drugs. Intermediate levels of MDRl mRNA are found in normal lung tissue. P-gp expression by epithelial cells lining bronchi has been demonstrated (Van der Valk et al., 1990). By slot-blot analysis, low MDRl mRNA levels were detected in 709 NSCLC tumors, but absent in 19 SCLC lines and one fresh tumor sample (Goldstein et al., 1989). In another study based on slot-blot analysis (Lai et al., 1989), 24 lung cancers of various pathology were studied for MDRl mRNA. Intermediate range mRNA levels, by reference to the multidrug-resistant line KB-8-5, were present in 3/6 SCLC and 8/13 NSCLC but were not significantly different from paired non-tumorous lung tissue in 10 cases with normal lung available for study. Higher levels were observed in one case of NSCLC with neuroendocrine markers. However, by PCR, MDRl transcripts were detectable in 16/18 lung cancers of unspecified pathology (Noonan et al., 1990). In a series of untreated lung carcinoma samples, mRNA levels were measurable by PCR in 4/8 SCLC samples, 13/16 adenocarcinoma of lung with high levels in six samples and in 3/4 other NSCLC samples (Holzmayer et al., 1992).

Chapter 3

Modulation

of P-Glycoprotein

Rationale and Strategies In order to determine if MDRlIP-gp is a potential target for reversing or circumventing clinical drug resistance, it is important to study diseases in which the overexpression of P-gp is believed to be an important factor in determining therapeutic outcome. To date, the most convincing evidence that P-gp plays a role in conferring clinical resistance has been in studies of hematopoietic malignancies and certain childhood malignarrcies (Dalton et al., 1991; Chan et al., 1991). As described in the previous chapter, Chan et al. have used immunohistochemical techniques to detect P-gp in childhood soft tissue sarcomas and neuroblastomas (Chan et al., 1990, 1991). In both of these childhood malignancies, the expression of P-gp was found to be an important adverse prognostic factor associated with drug resistance and poor outcome. MDRlIP-gp has also been consistently reported to be a predictor of poor outcome in patients with acute myeloblastic ieukemia (Pirker et al., 1991; Marie et al., 1991; Campos et al., 1992), non-Hodgkin’s lymphoma (Chabner et al., 1994; Miller et al., 1994) and multiple myeloma (Dalton et al., 1989; Epstein et al., 1989; Grogan et al., 1993). Based on these findings, it would appear that these malignancies are the most suitable for studies to reverse drug resistance. Means of overcoming clinical MDR might include the following possibilities: (1) the use of high close chemotherapy, as would be used in autologous bone-marrow transplants; (2) the development of new chemotherapeutic agents, which are not substrates for P-gp; (3) inhibiting the expression of the MDRl gene; and (4) the use of agents that are capable of inhibiting the function of P-gp in tumor cells. To date, the area that has received the most attention, both in the laboratory and the clinic, is the use of agents to reverse P-gp function. The concept of reversing MDR with antagonists of P-gp function,, termed chemosensitizers, is based on the seminal observation of Tsuruo and co-workers, who noted that the calcium-channel blocker, verapamil, blocked vincristine resistance in vitro and in vivo using a murine leukemia model (Tsuruo et al., 1982b). Although calcium-channel blockers as a class of reagents are very effective in inhibiting P-gp function, calcium ion movement appears to have nothing to do with this activity (Naito and Tsuruo, 1989). Since the original observation of Tsuruo et al., a number of structurally diverse agents have been found to reverse MDR (see Table 2) (Beck, 43

44

C. Shustik et al. Table 2. Agents that inhibit P-glycoprotein Type/Chemical

class

function

Example

Calcium channel blockers Calmodulin inhibitors Coronary vasodilators Immunosuppressant Indole alkaloids Detergents Quinolines

Phenylalkylamines, e.g. verapamil Phenothiazines Dipyridamole, Amiodarone Cyclosporin A, FK506 Reserpine Tween 80 Quinine

1991). Studies of structure-activity relationships of chemosensitizers performed by Beck and colleagues have demonstrated that modulators of P-gp are generally lipid soluble at physiological pH and possess a basic nitrogen atom and two planar aromatic rings (Zamora et al., 1988; Pearce et al., 1989). Although the evidence is indirect, it appears that essentially all of the chemosensitizers are effective at binding P-gp, thereby inhibiting its function (Safa et al., 1987). Other possibilities as mechanisms for P-gp inhibition have been suggested, such as alterations in the phosphorylation state of P-gp; however, the supporting evidence for these alternative explanations is less convincing than that of competitive binding of P-gp (Hamada et al., 1987). to reverse MDRlIP-gp have used various to reverse MDR in pre-clinical models, but were not drugs originally designed for this purpose. These chemosensitizers may be classified as ‘first generation chemosensitizers’ and include racemic verapamil, cyclosporin A, trifluoperazine, tamoxifen, quinine and others (see Table 3). Because these agents were used for a novel purpose, the studies conducted to date have been Phase I and II clinical trials. The primary aim of these studies was to determine if adequate doses of chemosensitizing agents could be combined with cytotoxic drugs and to evaluate for any unusual toxicities. Target concentrations of chemosensitizing agents can theoretically be established by using pre-clinical models, but the effectiveness of such agents is likely to depend on a number of factors including: (1) the intrinsic activity of the agent to reverse P-gp; (2) the tumor type being treated; (3) the anatomical location of the tumor; and (4) the amount of P-gp expressed in the tumor cells. In the past decade, clinical trials conducted

chemosensitizing

agents,

which

were

known

Ultimately, as with most pharmacological agents, the maximum tolerated dose (MTD) of a chemosensitizing agent is determined by the toxicity observed in clinical trials. In conducting these studies, however, investigators must consider not only the toxicity inherent for the chemosensitizing agent, but also the possibility of enhancing the toxicity of the chemotherapeutic drug. Enhanced toxicity of the chemotherapeutic drugs by the chemosensitizer may result from at least two distinct mechanisms: (1) the inhibition of P-gp function in normal tissues, and/or (2) by altering the clearance of the chemotherapeutic drug. Considering the first possibility, studies using mice in which the mdr3 gene has been deleted (the ultimate in P-gp inhibition), have shown that drug distribution can be dramatically altered, resulting in increased concentration

P-Glycoprotein-Mediated

Multidrug

45

Resistance

Table 3. Clinical studies to reverse MDRlIP-gp Agent

Study type

Investigator

Racemic Verapamil

1. 2. 3. 4.

Phase I: Vlb + Verap Ovarian Cancer: Dox + Verap Solid Tumors: Dox + Verap Pediatric leukemia: Vlb + VP-16 + Verap 5. Myeloma/NHL: VAD + Verap 6. NHL: C-VAD + Verap 7. Myeloma: VAD + Verap

Benson (1985) 0~01s (1987) Presant (1986) Cairo (1989)

Trifluoperazine

1. Phase I/II: Dox + Trifluo

Miller (1988)

Quinine

1. AML: Ara-ClMitox

Solary (1992)

Quinidine

1. Breast cancer: Epi + Quinidine

Jones (1990)

Bepridil

1. Colorectal cancer: Vlb + Bepridil

Linn (1994)

Tamoxifen

1. Phase I: Vlb + Tam

Trump (1992)

Progesterone

1. Phase I: Dox + Prog

Christen (1993)

Cyclosporin A

1. 2. 3. 4. 5. 6. 7.

+ .Quinine

Phase I: VP-16 + CsA Phase I: Vlb + CsA Phase I: Dox + CsA Phase I: Dox + CsA AML: Ara-C/Daun + CsA AML: MitoxNP-16 + CsA Myeloma: VAD + CsA

Dalton (1989) Miller (1991) Salmon (1991)

Lum (1992) Samuels (1993) Erlichman (1993) Bartlett (1994) List (1992) Marie (1993) Sonneveld (1992)

of cytotoxic drug into tissues normally protected by P-gp (e.g. the brain) and that this alteration leads to enhanced toxicity. In the second case, an alteration in the clearance

of the cytotoxic drug by the chemosensitizer would result in toxicity analo.gous to an increase in the dose of the cytotoxic drug (Schinkel et al., 1994). Clinical evidence for the later possibility has been presented by Sikic and co-workers, for the combination of cyclosporin A and etoposide (Lum et al., 1992, 1993), and Samuels et al. for cyclosporin A and vinblastine (Samuels et al., 1993). A third possibility resulting in unique toxicities associated with chemosensitizers plus cytotoxic agents, is a combination of the two possibilities noted above; in other words, the inhibition of normal P-gp function in an organ involved in eliminating the cytotoxic drug, such as liver or kidney. Such a scenario would rlesult in altered clearance of the cytotoxic drug and enhanced toxicity. These studies (demonstrate that doses of the chemosensitizer and cytotoxic drugs in clinical studies must be adjusted to provide optimal concentrations for reversing resistance, but at the same time to avoid undue toxicity.

46

Clinical Trials to Modulate

C. Shustik et al.

Multidrug

Resistance

The accumulating evidence for the expression of P-gp in tumors with intrinsic or acquired resistance to chemotherapy led to the early clinical investigation of noncytotoxic agents, which could competitively inhibit P-gp function in in vitro multidrugresistant cell lines. Verapamil, a calcium-channel blocker, which had been shown to enhance cytotoxicity of Vinca alkaloids in vincristine-resistant P388 leukemia (Tsuruo et al., 1981), was the first drug to be tested as a modulator of drug resistance in a clinical setting. An early trial (0~01s et al., 1987) of verapamil administered with doxorubicin to eight patients with drug-resistant ovarian carcinoma was negative, but in this study there was no evaluation of P-gp expression in tumor specimens. A negative Phase II trial was subsequently reported using verapamil and mitoxantrone in advanced ovarian carcinoma (Hendrick et al., 1991). Initial positive results with this drug were reported in pilot studies of myeloma and lymphoma (Dalton et al., 1989). These investigators observed a transient response to VAD with the addition of continuous infusion verapamil in three out of six patients (two with myeloma, one with NHL) with P-gp-positive tumors who developed progressive disease during treatment with a regimen containing vincristine and doxorubicin. These observations were extended in a subsequent study of verapamil as a chemosensitizer in 18 patients with Hodgkin’s disease and NHL failing on therapy or relapsing within 3 months of treatment with a doxorubicin/vincristine containing regimen (Miller et al., 1991). P-gp expression defined by immunohistochemical staining of >30% of malignant cells was found in 7/11 biopsies available for study before treatment. Using a treatment regimen of cyclophosphamide, continuous infusion doxorubicin and vincristine, and dexamethasone (CVAD) along with verapamil to maximally tolerated doses, complete and partial responses were achieved in 13/18 (72%) patients including 5/7 with P-gp-positive tumors. However, responses were also observed in 2/4 patients without detectable P-gp expression and the contribution of verapamil to the efficacy of CVAD in this study has been questioned (Chabner and Wilson et al., 1991). In a larger trial in patients with multiple myeloma refractory to VAD, infusional verapamil was administered with VAD to 22 patients with disease progression during VAD therapy (Salmon et al., 1991). MDRl expression in marrow plasma cells was determined by immunohistochemical staining with C219 in 15 patients prior to treatment. Partial responses of brief duration were achieved in 5/22 patients with 4/10 responders among the group with MDRl-positive myeloma cells, but no responses in five patients with MDRI-negative myeloma. As in other studies, cardiovascular toxicity including hypotension, fluid retention and conduction abnormalities was dose-limiting for verapamil. A randomized Phase II study of verapamil with single agent epirubicin was conducted in advanced metastatic breast cancer (Mross et al., 1993). Fifty-one patients were treated with epirubicin by i.v. bolus over 3 days with or without a daily dose of 480 mg verapamil of orally administered one day before and during epirubicin administration. In 48 evaluable patients, the objective response rate was approximately 30% in both groups with comparable overall survival, but P-gp determinations in tumor specimens were not performed. Continuous infusion verapamil was tested in combination with bolus vinblastine and continuous infusion VP-16 (Cairo et al., 1989) in pediatric patients with refractory leukemia (5), neuroblastoma (1) and hepatoblastoma (1). All patients had been heavily pre-treated with MDR-related drugs, but MDR tumor status was not evaluated. Partial responses, primarily cytoreduction of peripheral blood leukemic blasts, were achieved following

P-Glycoprotein-Mediated

S/11treatment

were attained, resistance.

Multidrug

Resistance

47

courses. Steady-state concentrations of approximately 1 PM verapamil which corresponds to levels required to reverse in vitro multidrug

The cardiovascular toxicity of verapamil limits the administration of doses required to achieve concentrations necessary for complete reversal of MDR in resistant cell lines in vitro and has diverted interest to the R-isomer of verapamil, which is less potent as a calcium-channel blocker and is associated with less cardiotoxicity, however, has an MDR-Imodulating potential comparable to the S-isomer or the racemic mixture. In a Phase I trial with a crossover design, in which R-verapamil was administered with EPOCH chemotherapy to patients with stable or progressive disease with EPOCH alone, objective responses were achieved in 5/21 (24%) of patients treated. Dose escalation of R-verapamil to levels required for reversal of in vitro resistance was possible in some patients with modest hypotension as a side-effect (Chabner et al., 1994). Cyclosporin A, a cyclic endecapeptide, which has wide clinical application as an immunosuppressive agent, was shown to competitively inhibit the efflux activity of P-gp (Slater et al., 1986) in leukemic cells in vitro. Further testing confirmed the potent in vitro inhibitory effect of cyclosporin A on P-gp function at concentrations, which could be achieved clinically (Ford and Hait, 1990). The modulation of MDR by cyclosporin A was studied in 21 patients with multiple myeloma (Sonneveld et al., 1992). Patients selected for this protocol included six unresponsive to primary therapy and 15 resistant to or progressing on VAD. Cyclosporin A was administered as an initial bolus before treatment with VAD and then by continuous infusion at 5 mg/kg, 7.5 mg/kg and 10 mg/kg daily during VAD treatment. P-gp expression in plasma cells was assayed by immunohistochemical staining with C219 and JSBl using an APAAP technique in 15 patients before treatment. The overall response rate was 48% (10/22) with seven responders among patients refractory to VAD. Responses were obs’erved in 7/12 (58%) of patients with plasma cell P-gp expression and, in 6/8 MDRZ-positive patients with evaluable pre-treatment and post-treatment samples, MDRI-positive plasma cells became undetectable after three cycles of chemotherapy. No respcmses were observed in the three MDRI-negative patients. Cyclosporin A steady-state concentrations of 800-1000 rig/l required for in vitro MDR modulation were attained at continuous infusion doses of 7.5 and 10 mg/kg daily. In a Phase I trial to determine the maximum tolerated dose (MTD) of continuous infusion cyclosporin A as an MDR modulator, 57 patients with assorted malignancies were treated with etoposide (VP-16) and cyclosporin A (Yahanda et al., 1992). Partial and minor responses were observed in four patients (one Hodgkin’s, one NHL, two ovarian carcinoma) refractory to etoposide. Steady-state levels were variable between patients, but at doses >15 mg/kg!day, levels greater than 2000 rig/l were achieved in 91% of cycles. Dose-related reversible hyperbilirubinemia due to inhibition of bilirubin transport was observed in 78% of courses with these levels. Hypomagnesemia, hypertension and impairment of renal function were associated with cyclosporin A, and enhanced myelosuppression and nausea with the concomitant administration of cyclosporin A and etoposide. The administration of cyclosporin A at doses resulting in steady-state plasma concentrations >2000 rig/l markedly altered etoposide pharma-

48

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cokinetics, consistent with inhibition of P-gp-mediated drug transport by proximal tubular epithelium and biliary epithelium as well as by a possible effect on drug metabolism. The resultant area-under-the-curve (AUC) of etoposide increased by 80%, with a greater than two-fold increase in plasma half-life and as a consequence, more profound neutropenia then occurred following the same dose of etoposide alone (Lum et al., 1992). A similar effect of cyclosporin A on the pharmacokinetics of doxorubicin was observed in a Phase I study (Bartlett et al., 1994). Paired pharmacokinetic studies in 12 patients treated with doxorubicin alone and the combination of cyclosporin A and doxorubicin demonstrated an increase in the plasma AUC of doxorubicin and, strikingly, of its less cytotoxic metabolite, doxorubicinol, by the concomitant administration of cyclosporin A. Impairment of the heptitic drug metabolism system, including NADPH-cytochrome-c reductase and cytochrome P-450-dependent hydroxylation and demethylation, by cyclosporin A is postulated as the mechanism for decreased metabolism of doxorubicinol. A dose reduction of doxorubicin to 60% when given with cyclosporin A resulted in neutropenia equivalent to 100% dose of doxorubicin alone. The potential importance of altered cytotoxic pharmacokinetics as a consequence of the administration of an MDR-modulating agent is underscored by the results of a Phase I/II trial of cyclosporin A in patients with poor-risk acute myeloid leukemia treated with cytosine arabinoside and daunorubicin (List et al., 1993). Leukemic cell expression of P-gp was determined by flow cytometry and immunocytochemistry using the MoAbs JSB-1, C219, C494 and MRK16, and MDRl mRNA was quantitated by a PCR assay. Forty-two patients with AML associated with adverse prognostic factors (relapse or primary refractory, post-cytotoxic or antecedent myelodysplasia, blast transformation of CML, or adverse cytogenetic abnormalities) were treated with 3 gm/m2 cytosine arabinoside daily for 5 days followed by 45 mg/m2 daunomycin by continuous infusion for 3 days with concomitant cyclosporin A by continuous infusion after a loading dose. Complete responses (including return to chronic phase CML) were achieved in 26/42 (62%) patients, but were unaffected by leukemic MDR phenotype. The response rate, rather, was higher in patients developing hyperbilirubinemia and in whom mean plasma daunorubicin levels were elevated. Considerations

for Future Clinical Trials of MDR Modulators

Studies conducted in patients with hematopoietic malignancies have been the most promising in demonstrating the potential of reversing clinical MDR. Studies using high dose infusion of verapamil in combination with vincristine/doxorubicin/dexamethasone (VAD) have been encouraging in demonstrating possible reversal of MDR in patients with myeloma who had progressed on VAD alone. However, responses in these patients were short lived and toxicity due to verapamil was high (Dalton et al., 1989; Salmon et al., 1991; Pennock et al., 1991). More recent studies using high dose infusion cyclosporin A (CsA) as a chemosensitizer, have shown promising results in patients with drug-resistant myeloma and AML (Sonneveld et al., 1992; List et al., 1993; Marie et al., 1993). In several studies, the number of P-gp-positive cells were reduced in patients with drug-resistant disease after the treatment with the CsAlcytotoxic drug combination. These studies suggest that CsA as a chemosensitizer is effective in eliminating P-gp-expressing malignant cells. Interpreting results of these studies is difficult, however, because of the findings of Sikic and co-workers and others who have demonstrated that CsA alters the clearance of certain cytotoxic agents and increases

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the dose intensity of the cytotoxic agents (Lum et al., 1992, 1993; Samuels et al., 1993). Future clinical studies must exclude the possibility that the improved response observed with the addition of chemosensitizing agents is due to increased cytotoxic drug exposure. One of the most important lessons learned from the Phase I and II trials conducted to date, is that the first generation chemosensitizers are not ideal for reversing MDR in the clinic. This conclusion is based on the observation that it is difficult to obtain adequate concentrations of the chemosensitizer in patients without prohibitive toxicity, due to the pharmacokinetic interactions between chemosensitizers and cytotoxic drugs. Given these observations, the characteristics of the ‘ideal’ chemosensitizer would probably be the following: (1) high level of efficacy with complete inhibition of P-gp function at low concentration - this characteristic would increase the possibility that adequate levels of the chemosensitizer could be obtained in patients; (2) no pharmacokinetic interaction with cytotoxic drugs - this would decrease the probability of increased toxicity due to increased exposure to cytotoxic drugs; and (3) selective inhibition of P-gp function in tumors while sparing P-gp function in normal tissues - this characteristic would reduce the probability of producing novel toxicities due to altered cytotoxic drug distribution. It is clear that more effective and less toxic agents are necessary in clinical trials aiming to reverse MDR. Design of future clinical trials using chemosensitizers will depend on the desired end point or objective. If the objective is to determine if chemosensitizers improve overall survival a.nd reduce mortality, then only a Phase III randomized trial will answer this question. This type of study will require a large number of patients as well as long-term follow-up. The question of pharmacokinetic interaction between the chemosensitizer and cytotoxic drugs is also difficult to address in this type of study design. An alternative design to study the effectiveness of the chemosensitizer is a two-stage trial design (chemotherapy until progression followed by the same chemotherapy plus chemosensitizer) . This type of design has been used in studying myeloma and malignant lymphomas (Chabner et al., 1994; Salmon et al., 1991; Sonneveld et al., 1992; Dalton, 1993). A reduction in tumor size, if it occurs, must be attributable to the addition of the chemosensitizer. This strategy, with the patient serving as his own control, allows for correlative analysis, such as the detection and elimination of P-gp-positive cells, and pharmacokinetic analysis for possible drug interactions. If results from a two-stage design Phase II trial are promising, then a randomized Phase III trial is warranted. The performance of clinical trials to reverse MDR will be critical to answering the question, “is MDRlIP-gp expression relevant to therapeutic outcome in cancer?” In addressing this question, three potential outcomes of these studies may be considered: Possibility 1: Clinical MDR can be reversed and therapeutic the use alf chemosensitizers.

outcome is improved by

Possibility 2: Clinical MDR cannot be reversed by available chemosensitizing agents. This would be demonstrated by detecting P-gp-positive tumor cells in patients who have been treated with chemosensitizers plus chemotherapy.

C. Shustik et al.

50

Table 4. Possible reasons for the failure of chemosensitizers 1. 2. 3. 4.

to reverse clinical MDRIIP-gp

Inability of chemosensitizer to achieve effective concentration at tumor site. Increase in P-gp expression as tumor progresses. Mutation in MDRIIP-gp that reduces the binding of chemosensitizers to P-gp. Alternative, non-P-gp mechanisms of drug resistance emerge that are not reversed by chemosensitizers.

3: Clinical MDR is reversed by chemosensitizers, but therapeutic outcome is not improved. This possibility would be demonstrated by converting tumors that are P-gp-positive before treatment with chemosensitizers to tumors that are P-gp negative. In this scenario, the goal of eliminating MDRlFgp would have been achieved, but patients would not have obtained therapeutic benefit, presumably because of the emergence of non-P-gp-mediated drug resistance. Possibility

Reasons for the possible failure of chemosensitizers to reverse clinical MDR are shown in Table 4. The first three reasons are probable explanations for the lack of response as might be observed in the clinical situation described by Possibility 2 above. Studies using first generation agents, such as verapamil, are likely to fail in some circumstances because of the inability of the drug to reach a critical concentration at the tumor site. It is also possible that some chemosensitizing agents may actually induce P-gp expression, and tumors treated with cytotoxic agents plus chemosensitizers may have higher levels of P-gp expression following treatment. Site-directed mutagenesis studies of the MDRl gene have demonstrated that certain mutations can affect the efficacy of chemosensitizing agents, presumably by altering the binding of these agents to P-gp (Kajiji et al., 1994). The fourth reason for failure of chemosensitizers to reverse clinical MDR might be observed in the clinical situation described by Possibility 3. Alternative mechanisms of resistance that might emerge following treatment with chemosensitizers include alterations in the enzyme topoisomerase II, or the overexpression of proteins similar to P-gp, such as the MRP gene (Beck et al., 1987; Cole et al., 1992). The MRP protein is relatively unaffected by chemosensitizing agents known to inhibit P-gp. In summary, the identification of MDRlfP-gp as a mechanism of resistance to multiple chemotherapeutic drugs creates a whole new approach to the treatment of cancer patients. Diagnostic tests are now available to determine the prevalence of this drug resistance gene in tumors. The results to date demonstrate that the expression of MDRlIP-gp correlates with the emergence of drug resistance in certain hematopoietic malignancies and childhood tumors. Given these findings, it is reasonable to try to prevent or circumvent this form of drug resistance in the clinic. The use of chemosensitizing agents to inhibit the function of P-gp has received a great deal of attention in both the laboratory and the clinic. Several studies using first generation chemosensitizing agents, such as verapamil and cyclosporin A, have reported encouraging results in reversing MDR. However, several confounding variables have been identified, which makes interpretation of these results difficult. These variables include the following: (1) a working definition of clinical drug resistance (proven resistance to prior therapy before the chemosensitizer is added), which is critical

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in identifying the appropriate patient population for study; (2) the pharmacokinetic consequences of potential drug interactions between the chemosensitizing agent and cytotoxic drugs; and (3) the simultaneous existence of non-P-gp mechanisms of drug resistance, which are unaffected by chemosensitizers to reverse P-gp. Future study design and interpretation of results must attempt to control for these potential complicating factors. The development of new, more effective and less toxic chemosensitizing agents will help to determine if targeting the MDRl gene in patients is clinically feasible.

References

Abraham, E. H., Prat, A. G., Gerweck, L., Seneveratne, T., Arceci, R. J., Kramer, R., Guidotti, G. and Cantiello, H. F. (1993). The multidrug resistance (m&l) gene product functions as an ATP channel. Proc. Natl. Acad. Sci. U.S.A. 90, 312-316. Akiyama., S. I., Cornwell, M. M., Kuwano, M., Pastan, I. and Gottesman, M. M. (19$8). Most drugs that reverse multidrug resistance also inhibit photoaffinity labeling of P-glycoprotein by a vinblastine analog. Mol. Pharmacol. 33, 144-147. Al-Shawi, M. K. and Senior, A. E. (1993). Characterization phosphatase activity of Chinese hamster P-glycoprotein.

of the adenosine triJ. Biol. Chem. 268,

4197-4206.

Ambudkar, S. V., Lelong, I. H., Zhang, J., Cardarelli, C. O., Gottesman, M. M. and Pastan, I. (1992). Partial purification and reconstitution of the human multidrug res:istance pump: characterization of the drug stimulatable ATP hydrolysis. Proc. Na,rl. Acad. Sci. U.S.A.

89, 8472-8476.

Ames, G. F.-L. (1986). Bacterial periplasmic transport systems: Structure, and evolution. Annu. Rev. Biochem. 55, 397-42.5.

mechanism

Arceci, R. J., Croop, J., Horwitz, S. B. and Housman, D. E. (1988). The gene encoding multidrug resistance is induced and expressed at high levels during pregnancy in the secretory epithelium of the uterus. Proc. Natl. Acad. Sci. U.S.A. 85, 4350-4354.

Arceci, R. J., Stieglitz, K., Bras, J., Schinkel, A., Baas, F. and Croop, J. (1993). Monoclonal antibody to an external epitope of the human mdrl P-glycoprotein. Cancer Res. 53, 310-317.

Azzaria, M., Schurr, E. and Gros, P. (1989). Discrete mutations introduced in the predicted nucleotide-binding sites of the mdrl gene abolish its ability to confer multidrug resistance. Mol. Cell Biol. 9, 5289-5297. 53

C. Shustiket al.

54

Baer, M. R. and Bloomfield, C. D. (1991). Multidrug resistance leukemia. J. Natl. Cancer Inst. 83, 663-665.

in acute myeloid

Bartlett, N. L., Lum, B. L., Fisher, G. A., Brophy, N. A., Ehsan, M. N., Halsey, J. and Sikic, B. I. (1994). Phase I trial of doxorubicin with cyclosporine as a modulator of multidrug resistance. J. Clin. Oncol. 12, 835-842. Beth-Hansen, N. T., Till J. E. and Ling V. (1976). Pleiotropic phenotype of colchicineresistant CHO cells: cross resistance and collateral sensitivity. J. Cell Z’hysiol. 88, 23-32.

Beck, W. T. (1991). Modulators of P-glycoprotein

associated multidrug resistance. In: Drug Resistance, (R. F. Ozols, ed.), pp. 151-170. Kluwer Academic Publishers, Philadelphia.

Molecular

and Clinical Advances

in Anticancer

Beck, W. T. and Danks, M. K. (1991). Characteristics of multidrug resistance in human tumor cells. In: Molecular and Cellular Biology of Multidrug Resistance in Tumor Cells, (I. B. Roninson, ed.), pp. 3-55. Plenum, New York. Beck, W. T., Cirtain, M. C., Danks, M. K., Felsted, R. L., Safa, A. R., Wolverton, J. S. and Trent, J. M. (1987). Pharmacological, molecular, and cytogenetic analysis of ‘atypical’ multidrug-resistant human leukemic cells. Cancer Res. 47, 5455-5460.

Bell, D. R., Gerlach, J. H., Kartner, N., Buick, R. N. and Ling, V. (1985). Detection of P-glycoprotein in ovarian cancer: A molecular marker associated with multidrug resistance. J. Clin. Oncol. 3, 311-315. Benson, A. B., Trump, D. L., Koeller, J. M., Egorin, M. I., Olman, E. A., Witte, R. S. and Tormey, D. C. (1985). Phase I study of vinblastine and verapamil given by concurrent iv infusion. Cancer Treat. Rep. 69, 795-799. Berr,

F., Meier, P. J. and Stieger, B. (1993). Evidence for the presence of a phosphatidyl-choline translocator in isolated rat liver canalicular plasma membrane vesicles. J. Biol. Chem. 26, 3976-3979.

Bibi, E. and Beja, 0. (1994). Membrane topology of multidrug resistance protein expressed in Escherichia coli-N terminal domain. J. Biol. Chem. 269,19910-19915. Biedler, J. L. and Riehm, H. (1970). Cellular resistance to actinomycin D in Chinese hamster cells in vitro: Cross resistance, radioautoradiographic, and cytogenetic studies. Cancer Res. 30, 1174-1184. Lemerle, J. and Riou, G. Bourhis, J., Benard, J., Hartmann, O., Boccon-Gibod, (1989a). Correlation of MDRl Gene Expression with Chemotherapy in Neuroblastoma. J. Natl. Cancer Inst. 81, 1401-1405.

P-Glycoprotein-Mediated

Multidrug

Resistance

55

Bourhis, J., Goldstein, L. J., Riou, G., Pastan, I. and Gottesman, M. M. (1989b). Expression of a human multidrug resistance gene in ovarian carcinomas. Cancer Res. 49, 5062-5065. Boyer, J. L., Graf, J. and Meier, P. J. (1992). Hepatic transport systems regulating pHi, cell volume, and bile secretion. Annu. Rev. Physiol. 54, 415-438. Bradley, G., Georges, E. and Ling, V. (1990). Sex dependent and independent expression of the P-glycoprotein isoforms in Chinese hamster. J. Cell Physiol. 145, 39&408.

Bruggemann, E. P., Germann, U. A., Gottesman, M. M. and Pastan, I. (1989). Two different regions of phosphoglycoprotein are photoaffinity-labeled by azidopine. J. Biol. Chem. 264, 15483-15488.

Bruggemann, E. P., Currier, S. P., Gottesman, M. M. and Pastan, I. (1992). Characterization of the azidopine and vinblastine binding site of P-glycoprotein. J. Biol. Chem. 267,21020-21026. Buschman, E. and Gros, P. (1991). Functional analysis of chimeric genes obtained by exchanging homologous domains of the mouse mdrl and mdr2 genes. Mol. Cell Bier’. 11, 595-603.

Buschman, E. and Gros, P. (1994) The inability of the mouse mdr2 gene to confer multidrug resistance is linked to reduced drug binding to the protein. Cancer Res. 54, 4892-4898.

Buschman, E., Arceci, R. J., Croop, J. M., Che, M., Arias, I. M., Housman, D. E. and Gros, P. (1992). Mouse mdr2 encodes P-glycoprotein expressed in the bile canalicular membrane as determined by isoform specific antibodies. J. Biol. Chem. 267, 18093-18099. Cairo, M. S., Siegel, S., Anas, N. and Sender, L. (1989). Clinical trial of continuous infusion verapamil, bolus vinblastine, and continuous infusion VP-16 in drugresistant pediatric tumors. Cancer Res. 49, 1063-1066. Campos, L., Guyotat, D., Archimbaud, E., Calmard-Oriol, P., Tsuruo, T., Troncy, J., Treille, D. and Fiere, D. (1992). Clinical significance of multidrug resistance P-glycoprotein expression on acute nonlymphoblastic leukemia cells at diagnosis. Blood 79, 473-476.

Cangelosi, G. A., Martinetti, G., Leigh, J. A., Lee, C. C., Theines, C. and Nester, E. W. (1989). Role of Agrobacterium tumefaciens ChvA protein in export of p-1, 2-glucan. J. Bacterial. 171, 1609-1615. Capecchi, M. R. (1989). Altering the genome by homologous 2441, 1288-1292

recombination.

Science

56

C. Shustik et al.

Chabner, B. A., Bates, S. E., Fojo, A. T., Spolyar, M. and Wilson, W. H. (1994). Drug resistance in adult lymphomas. Semis. Hemafol. 31, 7&87. Chan, H. S., Thorner, P. S., Haddad, G. and Ling, V. (1990). Immunohistochemical detection of P-glycoprotein: prognostic correlation in soft tissue sarcoma of childhood. J. C&n. Oncol. 8, 689-704. Chan, H. S., Haddad, G., Thorner, P. S., DeBoer, G., Lin, Y. P., Ondrusek, N., Yeger, H. and Ling, V. (1991). P-glycoprotein expression as a predictor of the outcome of therapy for neuroblastoma. N. En@. J. Med. 325, 1608-1614. Chan, J. -S. L., Haddad, G., Zheng, L., Thorn&, P. S., Bradley, G., Dalton, W. S. and Ling, V. (1994). A sensitive multilayer immunofluorescent method for flow cytometric detection of P-glycoprotein in myeloma and tumor cells. Lab. Invest. (in press). Chaudhary, P. M. and Roninson, I. B. (1991). Expression and activity of P-glycoprotein, a multidrug efflux pump, in human hematopoietic stem cells. Cell 66, 85-94. Chen, C. -J., Chin, J. E., Ueda, K., Clark, D. P., Pastan, I., Gottesman, M. M. and Roninson, I. B. (1986). Internal duplication and homology with bacterial transport proteins in the mdrl (P-glycoprotein) gene from multidrug resistant human cells. Cell 47, 381-389. Cheng, A. -L., Su, I. -J., Chen Chang, Y., Lee Chang T. and Wang, C-H.(1993). Expression of P-glycoprotein and glutathione-S-transferase in recurrent lymphomas: the possible role of Epstein-Barr virus, immunophenotypes, and other predisposing factors. J. Clin. Uncol. 11, 109-115. Cheng, S. H., Gregory, R. J. ,. Marshall, J., Paul, S., Souza, D. W., White, G. A., O’Riordan, C. R. and Smith, A. E. (1991). Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell 63, 827-834. Chevalier-Multon, M-C., Jacquemin-Sablon, A., Bessel&e, R., Husson, H-P. and Le Pecq, J-B. (1990). Comparative cytotoxicities of a series of ellipticine and olivacine derivatives on multidrug resistant cells of human and murine origins Anti-Cancer Drug Des. 5, 319-335. Chin, J. E., Soffir, R., Noonan, K. E., Choi, K. and Roninson, I. B. (1989). Structure and expression of the human MDR (P-glycoprotein) gene family. Mol. Cell Biol. 9, 3808-3820. Choi, K., Chen, C. J., Kriegler, M. and Roninson,

I. B. (1988). An altered pattern of cross-resistance in multidrug resistance human cells results from spontaneous mutations in the MDRl (P-glycoprotein) gene. Cell 53, 519-529.

P-Glycoprotein-Mediated

Multidrug

Resistance

57

Christen, R. D., McClay, E. F., Plaxe, S. C., Yen, S. S., Kim, S., Kirmani, S., Wilgus, L. L., Heath, D. D., Shalinsky, D. R., Freddo, J. L., Braly, P. S., O’Quigley , J. and Howell, S. B. (1993). Phase I/pharmacokinetic study of high-dose progesterone and doxorubicin. J. Clin. Oncol. 11, 2417-2426. Cole, S. P., Bhardwaj, G., Gerlach, J. H., Mackie, J. E., Grant, C. E., Almquist, K. C., Kurz, E. U., Duncan, A. M. and Deeley, R. G. (1992). Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line. Science 258, 1650-1654. Coleman, R. (1987). Biochemistry

of bile secretion. Biochem. .I. 244, 249-261.

Conter, V. and Beck, W. T. (1984). Acquisition of multiple drug resistance by CCRFCEM cells selected for different degrees of resistance to vinblastine. Cancer Treat. Rep. 68, 831-839. Cordon-Cardo, C., O’Brien, J. P., Casals, D., Rittman-Grauer, L., Biedler, J. L., Melamed, M. R. and Bertino, J. R. (1989). Multidrug-resistance gene (P-glycoprotein) is expressed by endothelial cells at the blood-brain barrier sites. Proc. Natl. Acad. Sci. U.S.A.

86, 695-698.

Cornelissen, J. J., Sonneveld, P., Schoester, M., Raaijmakers, H. IK., Dekker, A. W. and Lokhorst, H. M. (1994). response to vincristine, doxorubicin, and dexamethasone myeloma refractory to alkylating agents. 1. Clin. Oncol.

H. G. P., Nieuwenhuis, Expression and chemotherapy in multiple MDR-1

12, 115-119.

Cornwell, M. M., Safa, A. R., Felsted, R. L., Gottesman, M. M. and Pastan, I. (1986a). Membrane vesicles from multidrug resistant cancer cells contain a specific 15@-170 kDa protein detected by photoaffinity labelling. Proc. N&l. Acad. Sci. U.S.A.

83, 3847-3850.

Cornwell, M. M., Gottesman, M. M. and Pastan, I. (1986b). Increased vinblastine binding to membrane vesicles from multidrug resistant KB cells. J. Biol. Chem. 261. 7921-7928.

Cornwell, M. M., Tsuruo, T., Gottesman, M. M. and Pastan, I. (1987a). ATP-binding properties of P-glycoprotein from multidrug resistant KB cells. FASEB J. 1, 51-54. Cornwell, M. M., Pastan, I. and Gottesman, M. M. (1987b). Certain calcium channel blockers bind specifically to multidrug resistant human KB carcinoma membrane ves:icles and inhibit drug binding to P-glycoprotein. J. Biol. Chem. 262,2166-2170. Croop, J. M., Raymond, M., Haber, D., Devault, A., Arceci, R. J., Gros, P. and Housman, D. E. (1989). The three mouse multidrug-resistance genes are expressed in a tissue specific manner in normal mouse tissues. Mol. Cell Biol. 9, :1346-1350.

C. Shustik et al.

58

Cumber, P. M., Jacobs, A., Hoy, T., Fisher, J., Whittaker, J. A., Tsuruo, T. and Padua, R. A. (1990). Expression of the multiple drug resistance gene (MDRI) and epitope masking in chronic lymphatic leukemia. Br. J. Huematol. 76, 226-230. Dalton,

W. S. (1993). Overcoming

Principles

Rosenberg,

the multidrug-resistant phenotype. In: Cancer: (V. DeVita Jr, S. Hellman and S. A. eds), pp. 2655-2666. J. B. Lippincott, Philadelphia.

and Practice

of Oncology

Dalton, W. S. and Grogan, T. M. (1991). Does P-glycoprotein predict response to chemotherapy, and if so, is there a reliable way to detect it? J. Natl. Cancer Inst. 83, 80-81. Dalton, W. S., Grogan, T. M. and Miller, T. P. (1991). The role of P-glycoprotein in drug-resistant hematologic malignancies. In: Molecular and Clinical Advances in Anticancer Drug Resistance (R. F. Ozols, ed.), pp. 187-208. Kluwer Academic Publishers, Boston. Dalton, W. S., Durie, B. G. M., Alberts, D. S., Gerlach, J. H. and Cress, A. E. (1986). Characterization of a new drug-resistant human myeloma cell line that expresses P- glycoprotein. Cancer Res. 46, 5125-5130. Dalton, W. S., Grogan, T. M., Meltzer, P. S., Scheper, R. J., Durie, B. G. M., Taylor, C. W., Miller, T. P. and Salmon, S. E. (1989a). Drug-resistance in multiple myeloma and non-Hodgkin’s lymphoma: detection of P-glycoprotein and potential circumvention by addition of verapamil to chemotherapy. J. Clin. Oncol. 7, 415-424.

Dalton, W. S., Grogan, T. M., Rybski, J. A., Scheper, R. J., Richter, L., Kailey, J., Broxterman, H., Pinedo, H. M. and Salmon, S. E. (1989b). Immunohistochemical detection and quantitation of P-glycoprotein in multiple drug-resistant human myeloma cells: association with level of drug resistance and drug accumulation. Blood 73, 747-752.

Dano, K. (1973). Active outward transport of daunomycin tumor cells. Biochim. Biophys. Acta. 323, 466-483.

in resistant Ehrlich ascites

De Bruijn, M. H. L., Van Der Bliek, A. M., Biedler, J. L. and Borst, P. (1986). Differential amplification and disproportionate expression of five genes in three multidrug resistant Chinese hamster lung cell lines. Mol. Cell Biol. 6, 4717-4722. Denning, G. M., Anderson, M. P., Amara, J. F., Marshall, J., Smith, A. E. and Welsh, M. J. (1992a). Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature- sensitive. Nature 358, 761-764. Denning, G. M., Ostedgaard, L. S. and Welsh, M. J. (1992b). Abnormal localization of cystic fibrosis transmembrane conductance regulator in primary cultures of cystic fibrosis airway epithelia. J. Cell Biol. 118, 551-559.

P-Glycoprotein-Mediated

Multidrug Resistance

59

Devault, A.. and Gros, P. (1990). Two members of the mouse mdr gene family confer multidrug resistance with overlapping but distinct drug specificities. Mol. Cell Biol. 10,1652-1663. Devine, S. E., Hussain, A., Davide, J. P. and Melera, P. W. (1991). Full length and alternatively spliced pgpl transcripts in multidrug resistant Chinese hamster lung cells. J. Biol. Chem. 266, 4545-4555. Devine, S. E., Ling, V. and Melera, P. W. (1992). Amino acid substitutions in the sixth transmembrane domain of P-glycoprotein alter multidrug resistance. Proc. Nutl. Acad. Sci. U.S.A.

89, 4564-4568.

Dhir, R. and Gros, P. (1992). Functional analysis of chimeric proteins constructed by exchanging homologous domains of two P-glycoproteins conferring distinct drug resistance profiles. Biochemistry 31, 6103-6110 Dhir, R., Grizzuti, K., Kajiji, S. and Gros, P. (1993) Modulatory effects on substrate specificity of independent mutations at the Ser9391941 position in predicted transmembrane domain 11 of P-glycoproteins. Biochemistry 32, 9492-9499. Di Marco, A. (1978). Mechanism of action and mechanism of resistance to antineoplastic agents that bind DNA. In: Fundamentals in Cancer Chemotherapy Antibiotics Chemotherapy. 23, pp. 216-227. Karger, Basel. Dudler,

R. and Hertig,

C. (1992). Structure

of an mdr-like gene from Arubidopsis

thalicma. J. Biol. Chem. 267, 5882-5888.

Durie, B. G. M. and Dalton, W. S. (1988). Reversal of drug-resistance myeloma with verapamil. Br. J. Haematol. 68, 203-206.

in multiple

El Rouby, S., Thomas, A., Costin, D., Rosenberg, C. R., Potmesil, M., Silber, R. and Newcomb, E. W. (1993). p53 gene mutation in B-cell chronic lymphocytic leukemia is associated with drug resistance and is independent of MDRlIMDR3 gene expression. Blood 82, 3452-3459. Endicott, J. A. and Ling, V. (1989) The biochemistry of P-glycoprotein multidrug resistance. Annu. Rev. Biochem. 58, 137-171.

mediated

Epstein, J., Xiao, H. Q. and Oba, B. K. (1989). P-glycoprotein expression in plasmacell myeloma is associated with resistance to VAD. Blood 74, 913-917. Erlichman, C., Moore, M., Thiessen, J. J., Kerr, I. G., Walker, S., Goodman, P., Bjarnason, G., DeAngelis, C. and Bunting, P. (1993). Phase I pharmacokinetic study of cyclosporin A combined with doxorubicin. Cancer Res. 53, 4837-4842. Fairchild: C. R., Ivy, S. P., Kao, C. S., Whang-Peng, J., Rosen, N., Israel, M. A., Melera, P. W., Cowan, K. H. and Goldsmith, M. E. (1987). Isolation of amplified and overexpressed DNA sequences from adriamycin resistant human breast cancer cells. Cancer Res. 47, 5141-5148.

C. Shustik et al.

60

Felmlee, T., Pellett, S. and Welch, R. A. (1985). Nucleotide sequence of a chromosomal hemolysin. J. Bacterial. 163, 94-105. Fojo A., Akiyama, S., Gottesman, M. M. and Pastan, I. (1985a). Reduced drug accumulation in multiple drug-resistant human KB carcinoma cell lines. Cancer Res. 45, 3002-3007.

Fojo, A. T., Whang-Peng, J., Gottesman, M. M. and Pastan, I. (1985b). Amplification of DNA sequences in human multidrug resistant KB carcinoma cells. Proc. Natl. Acad. Sci. U.S.A.

82, 7661-7665.

Fojo, A., Ueda, K., Slamon, D. J., Poplack, D. G., Gottesman, M. M. and Pastan, I. (1987a). Expression of a multidrug-resistance gene in human tumors and tissues. Proc. Natl. Acad. Sci. U.S.A.

84, 265-269.

Fojo, A. T., Shen, D. -W., Mickley, L. A., Pastan, I. and Gottesman, M. M. (1987b). Intrinsic drug resistance in human kidney cancer is associated with expression of a human multidrug- resistance gene. J. Clin. Oncol. 5, 1922-1927. Ford, J. and Hait, W. N. (1990). Pharmacology of cancer. Pharmacol. Rev. 42, 155-199.

of drugs that alter multidrug resistance

Foote, S. J., Kyle, D. E., Martin, R. K, Oduola, A. M. J., 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 falciparum. Nature 345, 255-258.

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 P. falciparum.

Cell 57, 921-930.

Futscher, B. W., Blake, L. L., Gerlach, J. H., Grogan, T. M. and Dalton, W. S. (1993). Quantitative polymerase chain reaction analysis of mdrl mRNA in multiple myeloma cell lines and clinical specimens. Anal. Biochem. 213, 414-421. Georges, E., Bradley, G., Gariepy, J. and Ling, V. (1990). Detection of P-glycoprotein isoforms by gene-specific monoclonal antibodies. Proc. Natl. Acad. Sci. U.S. A. 87, 152-156.

Gerlach, J. H., Bell, D. R., Karakousis, C., Slocum, H. K., Kartner, N., Rustum, Y. M. and Baker, R. M. (1987). P-glycoprotein in human sarcoma: evidence for multidrug resistance. .J. C/in. Oncoi. 5, 1452-1460. Gill, D. R., Hyde, S. C., Higgins, C. F., Valverde, M. A., Mintenig, G. M. and Sepulveda, F. V. (1992). Separation of drug transport and chloride channel functions of the human multidrug resistance P-glycoprotein. Cell 71, 23-32.

P-Glycoprotein-Mediated

Multidrug

Resistance

61

Gilson, E., Higgins, C. F., Hofnung, M., Ames, G. F. L. and Nikaido, H. (1982). Extensive homology between membrane associated components of histidine and maltose transport systems of Salmonella typhimurium and Escherichia coli. J. Biol. Chem. 257, 9915-9918.

Goasguen, J. E., Dossot, J. -M., Fardel, O., Le Mee, F., Le Gall, E., Le Blay, R., Le Prise, P. Y., Chaperon, J. and Fauchet, R. (1993). Expression of the multidrug resistance-associated P-Glycoprotein (P-170) in 59 cases of de ltovo acute lymphoblastic leukemia: prognostic implications. Blood 81, 2394-2398. Goldstein, L. J., Galski, H., Fojo, A., Willingham, M., Lai, S. L., Gazdar, A., Green, A., ICrist, W., Brodeur, G. M., Lieber, M., Cossman, J., Gottesman, M. M. and Pastan, I. (1989). Expression of a multidrug resistance gene, in human cancers. J. Natl. Cancer Inst. 81, 116-124,

1989.

Goldstein, L. J., Fojo, A. T., Ueda, K., Crist, W., Green, A., Brodeur, G., Pastan, I. and Gottesman, M. M. (1990). Expression of the multidrug resistance, MDR I, gene in neuroblastomas. J. Clin. Oncol. 8, 128-136. Gottesma.n, M. M. and Pastan, I. (1993). Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu. Rev. Biochem. 62, 385-427. Greenberger, L. M. (1993). Major photoaffinity drug labeling sites for iodoaryl azidoprazosin in P-glycoprotein are within or immediately C-terminal to, transmernbrane domains 6 and 12. J. Biol. Chem. 268, 11417-11425. Greenberger, L. M., Yang, C. P. H., Gindin, E. and Horwitz, S. B. (1990). Photoaffinity probes for the al-adrenergic receptor and the calcium channel bind to a common domain in P- glycoprotein. J. Biol. Chem. 265, 4394-4401. Greenberger L. M., Lisanti, C. J., Silva, J. T. and Horwitz, S. B. (1991). Domain mapping of the photoaffinity drug binding sites in P-glycoprotein encoded by mouse mdrlb. J. Biol. Chem. 266, 20744-20751. Grogan, T., Dalton, W., Rybski, J., Spier, C., Meltzer, P., Richter, L., Gleason, M., Pindur, J., Cline, A., Scheper, R., Tsuruo, T. and Salmon, S. (1990). Optimization of immunocytochemical P-glycoprotein assessment in multidrugresistant plasma cell myeloma using three antibodies. Lab. Invest. 63, 815-824. Grogan, T. M., Spier, C. M., Salmon, S. E., Matzner, M., Rybski, J., Weinstein, R. S., Scheper, R. J. and Dalton, W. S. (1993). P-glycoprotein expression in human plasma cell myeloma: correlation with prior chemotherapy. Blood 81,490-495. Gros, P., Croop, J., Varshavsky, A. and Housman, D. E. (1986a). Isolation and characterization of DNA sequences amplified in multidrug resistant hamster cells. Proc. Natl. Acad. Sci. U.S.A.

83, 337-341.

62

C. Shustik et al.

Gros, P., Croop, J. M and Housman, D. E. (1986b). Mammalian multidrug resistance gene: complete cDNA sequence indicates strong homology to bacterial transport proteins. Cell 47, 371-380. Gros, P., Ben Neriah, Y., Croop, J. M. and Housman, D. E. (1986~). Isolation and expression of a complementary DNA that confers multidrug resistance. Nature 323, 728-73 1. Gros,

P., Raymond, M., Bell, J. and Housman, D. E. (1988). Cloning and characterization of a second member of the mouse mdr gene family. Mol. Cell Biol. 8, 2770-2778.

Gros, P., Dhir, R., Croop, J. M. and Talbot, F. (1991). A single amino acid substitution strongly modulates the activity and substrate specificity of the mouse mdrl and mdr3 drug efflux pumps. Proc. Natl. Acad. Sci. U.S.A. 88, 7289-7293. Grund, S. H., Patil, S. R., Shah, H. 0.) Pauw, P. G. and Stadler, J. K. (1983). Correlation of unstable cross-resistance in Chinese hamster ovary cells with a homogeneously staining region on chromosome 1. Mol. Cell Biol. 3, 1634-1647. Guild, B. C., Mulligan, R. C., Gros, P. and Housman, D. E. (1988). Retroviral transfer of a murine complementary DNA for multidrug resistance confers pleiotropic drug resistance to cells without prior drug selection. Proc. N&l. Acud. Sci. U.S.A. 85, 1595-1599. Gupta, R. S. (1983). Genetic, biochemical, and cross-resistance studies with mutants of Chinese hamster ovary cells resistant to anti-cancer drugs, VM-26 and VP-16-213. Cancer Res. 43, 1568-1574. Hamada, H., Hagiwara, K., Nakajima, T. and Tsuruo, T. (1987). Phosphorylation of the Mr 170,000 to 180,000 glycoprotein specific to multidrug-resistant tumor cells: effects of verapamil, trifluoperazine, and phorbol esters. Cancer Res. 47, 2860-2865. Hamada, H. and Tsuruo, T. (1986). Functional role for the 170- to 180-kDa glycoprotein specific to drug-resistant tumor cells as revealed by monoclonal antibodies. Proc. Natl, Acad. Sci. U.S. A. 83, 7785-7789. Hamada, H. and Tsuruo, T. (1988). Purification of the 170-to 180-kilodalton membrane glycoprotein associated with multidrug resistance. J. Biol. Chem. 263, 1454-1458. Hammond, J., Johnstone, R. M. and Gros, P. (1989). Enhanced efflux of [3H] vinblastine from Chinese hamster ovary cells transfected with a full length complementary DNA clone for the mdrl gene. Cancer Res. 49, 3867-3871. Henderson, D. M., Sifri, C. D., Rodgers, M., Wirth, D. F., Hendrickson, N. and Ullman, B. (1992). Multidrug resistance in Leishmaniu donovani is conferred by amplification of a gene homologous to the mammalian mdrl gene. Mol. Cell Biol. 12.2855-2865.

P-Glycoprotein-Mediated

Multidrug

63

Resistance

Hendrick, A. M., Harris, A. L. and Cantwell B. M. J. (1991). Verapamil with mitoxantrone for advanced ovarian cancer: A negative Phase II trial. Ann. Oncol. 2, 71-72. Herweijer., H., Sonneveld, P, Baas, F. and Nooter, K. (1990). Expression of mdrl and mdr3 multidrug-resistance genes in human acute and chronic leukemias and association with stimulation of drug accumulation by cyclosporine. J. N&l. Cancer Inst. 82, 1133-1140.

Herweijer, H., Nooter, K., Beishuizen, A., Sonneveld, P., Oostrum, R. G., HanssenHesseling, A. L. W. and van Dongen J. J. M. (1991). Expression of mdrl and mdr.3 multidrug-resistance genes in hairy cell leukaemia. Eur. J. Cancer 27, 297-298.

Higgins, C. F. (1992). ABC transporters: Cell Biol. 8, 67-113

from microorganisms

to man. Annu. Rev.

Higgins, C. F. (1994). To flip or not to flip? Curr. Biol. 4, 259-260. Higgins, C. F., Haag, P. D., Nikaido, K., Ardeshir, F., Garcia, G. and Ames, G. F. I,. (1982). Complete nucleotide sequence and identification of membrane components of the histidine transport operon of Salmonella typhimurium. Nature 298, 723-727.

Higgins, C. F., Hydes, I. D., Salmond, G. P. C., Gill, D. R., Downie, J. A., Evans, I. J., Holland, I. B., Gray, L., Buckel, S. D., Bell, A. W. and Hermondson, M. A. (1986). A family of related ATP-binding subunits coupled to many distinct biological processes in bacteria. Nature 323, 448-450. Holmes, IH., Jacobs, A., Carter, G., Janowska-Wieczorek, A. and Padua, R. A. (1989). Multidrug resistance in haemopoietic cell lines, myelodysplastic syndromes and acute myeloblastic leukaemia. Br. J. Huemutol. 72, 40-44. Holmes, .J. A., Jacobs, A., Carter, G., Whittaker, J. A., Bentley, D. P. and Padua, R. A. (1990) Is the mdrl gene relevant in chronic lymphocytic leukemia? Leukemia 4, 216-218.

Holzmayer, T. A., Hilsenbeck, S., Von Hoff, D. D. and Roninson, I. B. (1992). Clinical correlates of MDRl (P-glycoprotein) gene expression in ovarian and sma.ll-cell lung carcinomas. J. Nutl. Cancer Inst. 84, 1486-1491. Horio, M., Gottesman, M. M. and Pastan, I. (1988). ATP-dependent transport of vinblastine in vesicles from human multidrug-resistant cells. Proc. Null. Acad. Sci. U.S.A.

85, 3580-3584.

Howell, IV., Belli, T. A., Zaczkiewicz, L. T. and Bell, J. A. (1984). High level, unstable adr:iamycin resistance in a Chinese hamster mutant cell line with double minute chromosomes. Cancer Res. 44, 4023-4029.

64

C. Shustik ef al.

Hsu, S. I. H., Cohen, D., Kirschner, L. S., Lothstein, L., Hartstein, M. and Horwitz, S. B. (1990). Structural analysis of the mouse mdrla (P-glycoprotein) promoter reveals the basis for differential transcript heterogeneity in multidrug resistant 5774.2 cells. Mol. Cell Biol. 10, 3596-3606. Ito, Y., Tanimoto, M., Kumazawa, T., Okumura, M., Morishima, Y., Ohno, R. and Saito, H. (1989). Increased P-glycoprotein expression and multidrug-resistance gene (mdrl) amplification are infrequently found in fresh acute leukemia cells. Cancer 63, 1534-1538. Itsubo, M., Ishikawa, T., Toda, G. and Tanaka, M. (1994). Immunohistochemical study of expression and cellular localization of the multidrug resistance gene product P-glycoprotein in primary liver carcinoma. Cancer 73, 298-303. Jones, R. D., Kerr, D. J., Harnett, A. N., Rankin, E. M., Ray, S. and Kaye, S. B. (1990). A pilot study of quinidine and epirubicin in the treatment of advanced breast cancer. Br. J. Cancer 62, 133-135. Juliano, R. L. and Ling, V. (1976). A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim. Biophys. Acta. 455, 152-162. Kacinski, B. M., Yee, L. D. and Carter, D. (1989). Quantitation of tumor cell expression in the P-glycoprotein (mdr) gene in human breast carcinoma clinical specimens. Cancer Bull. 41, 44-48. Kajiji, S., Talbot, F., Grissuti, K., Van Dyke-Phillips, V., Safa, A. and Gros, P. (1993). Functional analysis of P-glycoprotein mutants identifies predicted trans-membrane 11 as a putative drug binding site. Biochemistry 32, 4185-4194. Kajiji, S., Dreslin, J., Grizzuti, K. and Gros, P. (1994). Structurally distinct MDR modulators show specific patterns of reversal against P-glycoproteins bearing unique mutations at serine939’941.Biochemistry 33, 5041-5048. Kakehi, Y., Kanamaru, H., Yoshida, O., Ohkubo, H., Nakanishi, S., Gottesman, M. M. and Pastan, I. (1988). Measurement of multidrug-resistance messenger RNA in urogenital cancers: elevated expression in renal cell carcinoma is associated with intrinsic drug resistance. J. Urology 139, 862-865. Kamijo, K., Taketani, S., Yokota, S., Osumi, T. and Hashimoto, T. (1990). The 70-kDa peroxisomal membrane protein is a member of the Mdr (P-glycoprotein)related ATP-binding protein superfamily. J. Biol. Chem. 265, 4534-4540. Kamimoto, Y., Gatmaitan, Z., Hsu, J. and Arias, I. M. (1989). The function of Gp170, the multidrug resistance gene product, in rat liver canalicular membrane vesicles. J. Biol. Chem. 264, 11693-11698. Kamiwatari, M., Nagata, Y., Kikuchi, H., Yoshimura, A., Sumizawa, T., Shudo, N., Sakoda, R., Seto, K. and Akiyama, S. I. (1989). Correlation between

P-Glycoprotein-Mediated

Multidrug Resistance

65

reversing of multidrug resistance and inhibiting of 3H-azidopine photolabeling of P-glycoprotein by newly-synthesized dihydropyridine analogues in a human cell liine. Cancer Res. 49, 319&3195. Kanamaru, H., Kakehi, Y., Yoshida, O., Nakanishi, S., Pastan, I. and Gottesman, M. M. Q989). MDRl RNA levels in human renal cell carcinomas: Correlation with grade and prediction of reversal of doxorubicin resistance by quinidine in tumor explants. J. Natl. Cancer Inst. 81, 844-849. Kartner, N., Evernden-Porelle, D., Bradley, G and Ling, V. (1985). Detection of P-glycoprotein in multidrug resistant cell lines by monoclonal antibodies. Nature 316, 820-823. Kelly, A., Powis, S. H., Kerr, L. -A., Mockridge, I., Elliott, T., Bastin, J., UchanskaZiegler, B., Ziegler, A., Trowsdale, J. and Townsend, A. (1992). Assembly and function of the two ABC transporter proteins encoded in the human major histocompatibility complex. Nature 355, 641-644. Klimecki, W. T., Futscher, B. W., Grogan, T. M. and Dalton, W. S. (1994). P-glycoprotein expression and function in circulating blood cells from normal volunteers. Blood 83, 2451-2458. Koch, G.: Smith, M., Twentyman, P. and Wright, K. (1986) Identification of a novel calcium-binding protein (CP22) in multidrug-resistant murine and hamster cells. FEBS

Lett. 195, 275-279.

Kuchler, K., Sterne, R. E. and Thorner, J. (1989). Saccharomyces cerevisiae STE6 gene product: a novel pathway for protein export in eukaryotic cells. EMBO 3.8, 3973-3984.

Kuwazuru, Y., Hanada, S., Furukawa, T., Yoshimura, A., Sumizawa, T., Utsunomiya, A., Ishibashi, K., Saito, T., Uozumi, K., Maruyama, M., Ishizawa, M., Arima, T. and Akiyama, S. I. (1990). Expression of P-glycoprotein in adult T-Cell leukemia cells. Blood 76, 2065-2071. Lai, S-L., Goldstein, L. J., Gottesman, M. M., Pastan, I., Tsai, C. M., Johnson, B. E., Mulshine, J. L., Ihde, D. C., Kayser, K. and Gazdar, A. F. (1989). MDRl Gene expression in lung cancer. J. Natl. Cancer Inst. 81, 1144-1150. Lemontt, J. F., Azzaria, M. and P. Gros. (1988). Increased MDR gene expression and decreased drug accumulation in multidrug-resistant human melanoma cells. Can’cer Res. 48, 6348-6353.

Lincke, C. R., The, I., van Groenigen, M and Borst P. (1992). The P-glycoprotein gene family of Caenorhabditis elegans: Cloning and characterization of genomic and complementary DNA sequences. J. Mol. Biol. 228, 701-711.

C. Shustik et al.

66

Ling, V. and Thompson, L. H. (1974) Reduced permeability in CHO cells as a mechanism of resistance to colchicine. J. Cell Physiol. 83, 103-116. Linn, S. C., van Kalken, C. K., van Tellingen, O., van der Valk, P., van Groeningen, C. J., Kuiper, C. M., Pinedo, H. M. and Giaccone, G. (1994). Clinical and pharmacologic study of multidrug resistance reversal with vinblastine and bepridil. J. Clin. Oncol. 12, 812-819. List, A. F., Spier, C. M., Cline, A., Doll, D. C., Garewal, H., Morgan, R. and Sandberg, A. A. (1991). Expression of the multidrug resistance gene product (P-glycoprotein) in myelodysplasia is associated with a stem cell phenotype. Br. J. Haematol. 78, 28-34.

List, A. F., Spier, C., Greer, J., Wolff, S., Hutter, J ., Dorr, R., Salmon, S., Baier, M. and Dalton, W. (1993). Phase I/II trial of cyclosporine as a chemotherapyresistance modifier in acute leukemia. J. Clin. Oncol. 11, 1652-1660. Loo, T. W. and Clarke, D. M. (1993a). Functional consequences in the predicted transmembrane domains of P-glycoprotein.

of proline mutations J. Biol. Chem. 268,

3143-3149.

Loo, T. W. and Clarke, D. M. (1993b). Functional consequences of phenylalanine mutations in the predicted transmembrane domains of P-glycoprotein. J. Biol. Chem. 268, 19965-19972.

Loo, T. W. and Clarke, D. M. (1994). Reconstitution of drug-stimulated ATPase activity following co-expression of each half of human P-glycoprotein as separate polypeptides. J. Biol. Chem. 269, 7750-7755. Ludescher, C., Hilbe, W., Eisterer, W., Thaler, J., Gotwald, M. and Hofmann, J. (1993a) Low incidence of activity of P-glycoprotein (P-170) in de novo acute lymphoblastic leukemia determined by flow cytometric assay. Blood 82, 3505-3506.

Ludescher, C., Wolfgang, H., Wolfgang, E., Preuss, E., Huber, C., Gotwald, M. and Hofmann, J. T. (1993b). Activity of P-glycoprotein in B-cell chronic lymphocytic leukemia determined by flow cytometric assay. J. Natl. Cancer Inst. 85,1751-1758. Lum, B. L., Kaubisch, S., Yahanda, A. M., Adler, K. M., Jew, L., Ehsan, M. N., Brophy, N.A., Halsey, J., Gosland, M. P. and Sikic, B. I. (1992). Alteration of etoposide pharmacokinetics and pharmacodynamics by cyclosporine in a Phase I trial to modulate multidrug resistance. J. Clin. Oncol. 10,1635-1642. Lum, B. L., Fisher, G. A., Brophy, N. A., Yahanda, A. M., Adler, K. M., Kaubisch, S. and Sikic, B. I. (1993). Clinical trials of modulation of multidrug resistance. Pharmacokinetic and pharmacodynamic considerations. Cancer 72, 3502-3514.

P-Glycoprotein-Mediated

Multidrug

Resistance

67

Ma, D. D. F., Davey, R. A., Harman, D. H., Isbister, J. P., Scurr, R. D.‘, Mackertich, S. M., Dowden, G. and Bell, D. R. (1987). Detection of multidrug resistant phenotype in acute non- lymphoblastic leukemia. Lancet 1987, 135-137. Marie, J. P., Zittoun, R. and Sikic, B. I. (1991). Multidrug resistance (m&l) gene expression in adult acute leukemias: correlations with treatment outcome and in vitro drug sensitivity. Blood 78, 586-592. Marie, J. I’., Bastie, J. N., Coloma, F., Faussat Suberville, A. M., Delmer, A., Rio, B., Delmas-Marsalet, B., Leroux, G., Casassus, P., Baumelou, E., Catalin, J. and Zittoun, R. (1993). Cyclosporin A as a modifier agent in the salvage treatment of acute leukemia (AL). Leukemia 7, 821-824. McGrath, J. P. and Varshavsky, A. (1989). The yeast STE6 gene encodes a homologue of the mammalian multidrug resistance P-glycoprotein. Nature 340, 400-404. Mechetner, E. B. and Roninson, I. B. (1992). Efficient inhibition of P-glycoproteinmediated multidrug resistance with a monoclonal antibody. Proc. Nutl. Acad. Sci. U.S.14. 89, 5824-5828. Merkel, D. E., Fugua, S. A. W., Tandona, A. K., Hill, S. M., Buzdar, A. U. and McGuire, W. L. (1989). Electrophoretic analysis of 248 clinical breast specimens for PGP overexpression or gene amplification. J. Clin. Oncol. 7, 1129-1136. Meyers, M. B., Spengler, B. A., Chang, T. D., Melera, P. W. and Biedler, J. L. (1985). Gene amplification-associated cytogenetic aberrations and protein changes in vincristine-resistant Chinese hamster, mouse, and human cells J. Cell Biol. 100, 588-597. Meyers, M. B., Shen, V., Spengler, B. A., Ciccarone, V., O’Brien, J. P., Donner, D. B., Furth, M. E. and Biedler, J. L. (1988). Increased epidermal growth factor receptor in multidrug-resistant human neuroblastoma cells. 1. Cell Biochem. 38, 87-97. Michieli, :M. G., Raspadori, A., Fanin, R., Fasalo, Baccarani, M. (1991). glycoprotein in B-cell 460-465.

D., Damiani, D., Geromin, A., Gallizia, C., Michelutti, G., Russo, D., Tazzari, P., Pileri, S., Mallardi, F. and The expression of the multidrug resistance-associated chronic lymphocytic leukaemia. Br. J. Huemutol. 77,

Miller, R. L., Bukowski, R. M., Budd, G. T., Purvis, J., Weick, J. K., Shepard, K. and Ganapathi, R. (1988). Clinical modulation of doxorubicin resistance by the calmodulin-inhibitor, trifluoperazine: a Phase I/II trial. J. Clin. Oncol. 6, 880-888. Miller, T. P., Grogan, T. M., Dalton, W. S., Spier, C. M., Scheper, R. J. and Salmon, S. E. (1991). P-glycoprotein expression in malignant lymphoma and reversal of clinical drug resistance with chemotherapy plus high-dose verapamil. 1. Clin. Oncol. 9, 17-24.

C. Shustik et al.

68

Miller, T. P., Chase, E. M., Dalton, W. S. and Grogan, T. M. (1994). The phenomenon of multidrug resistance in non-hodgkin’s lymphoma. In: Advances in Lymphoma Research, (F. Cabanillas and M. A. Rodriguez, eds), Kluwer Academic Publishers, Norwell. Miwa, H., Kita, K., Nishii, K., Morita, N., Takakura, N., Ohishi, K., Mahmud, N., Kageyama, S., Fukumoto, M. and Shirakawa, S. (1993). Expression of MDRl gene in acute leukemia cells: association with CD7+ acute myeloblastic leukemia/acute lymphoblastic leukemia. Blood 82, 3445-3451. Moscow, J. A., Fairchild, C. R., O’Brien, E. E., Poplack, D. (1989). Expression of anionic in human tissues and tumors.

Madden, M. J., Ranson, D. J., Wieand, H. S., G., Cossman, J., Myers, C. E. and Cowan, K. H. glutathione-S-transferase and P-glycoprotein genes Cancer Res. 49, 1422-1428.

Mosser, J., Douar, A. -M., Sarde, C. -0.) Kioschis, P., Feil, R., Moser, H., Poustka, A. -M., Mandel, J. -L. and Aubourg, P. (1993). Putative X-linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters, Nature 361,726-730. Mross, K., Bohn, C., Edler, L., Jonat, W., Queisser, W., Heidemann, E., Goebel, M. and Hossfeld, D. K. (1993). Randomized Phase II study of single-agent epirubicin +I- verapamil in patients with advanced metastatic breast cancer. Ann. Oncol. 4, 45-50. Musto, P., Melillo, L., Lombardi, G., Matera, R., Di Giorgio, G. and Carotenuto, M. (1991). High risk of early resistant relapse for leukaemic patients with presence of multidrug resistance associated P-glycoprotein positive cells in complete remission. Br. J. Haemutol. 77, 50-53. Naito,

M. and Tsuruo, T. (1989). Competitive inhibition by verapamil of ATPdependent high affinity vincristine binding to the plasma membrane of multidrugresistant K562 cells without calcium ion involvement. Cancer Res. 49, 1452-1455.

Nakamoto, R. K., Rao, R. and Slayman, C. W. (1991). Expression of the yeast plasma membrane [H+]ATPase in secretory vesicles. J. Biol. Chem. 266, 7940-7949. Ng, W. F., Sarangi, F., Zastawny, R. L., Veinot-Drebot, L. and Ling, V. (1989). Identification of members of the P-glycoprotein multigene family. Mol. Cell Biol. 9, 1224-1232.

Niehans, G. A., Jaszcz, W., Brunetto, V., Perri, R. J., Gajl-Peczalska, K., Wick, M. R., Tsuruo, T. and Bloomfield, C. D. (1992). Immunohistochemical identification of P-glycoprotein in previously untreated, diffuse large cell and immunoblastic lymphomas. Cancer Res. 52, 3768-3775.

P-Glycoprotein-Mediated

Multidrug Resistance

69

Noonan, K. E., Beck, C., Holzmayer, T. A., Chin, J. E., Wunder, J. S., Andrulis, I. L., Gazdar, A. F., Willman, C. L., Griffith, B., Von Hoff, D. D. and Roninson, I. B. (11990). Quantitative analysis of MDRl (multidrug resistance) gene expression in human tumors by polymerase chain reaction. Proc. Natl. Acad. Sci. U.S.A. 87, 7160-7164. Ouellette, M., Fase-Fowler, F. and Borst, P. (1990). The amplified H circle of methotrexate r resistant Leishmania tareqtolae contains a novel P-glycoprotein gene. EMBO J. 9, 1027-1033. Ozols, R. F., Cunnion, R. E., Klecker, R. W., Jr, Hamilton, T. C., Ostchega, Y. and Young, R. C. (1987). Verapamil and adriamycin in the treatment of drug-resistant ovarian cancer patients. J. Clin. Oncol. 5, 641-647. Pearce, H. L., Safa, A. R., Bach, N. J., Winter, M. A., Cirtain, M. C. and Beck, W. T. (11989). Essential features of the P-glycoprotein pharmacophore as defined by a series of reserpine analogs. Proc. Natl. Acad. Sci. U.S.A. 86, 5128-5132. Pennock, G. D., Dalton, W. S., Roeske, W. R., Appleton, C. P., Mosley, K., Plezia, P., Miller, T. P. and Salmon, S. E. (1991). Systemic toxic effects associated with high-dose verapamil infusion and chemotherapy administration. J. Natl. Cancer Inst. 83, 105-110. Peterson, R. H., Beutler, W. J. and Biedler, J. L. (1979). Ganglioside composition of malignant and actinomycin D resistant nonmalignant Chinese hamster cells. Biochem. Pharmacol. 28, 579-582. Peterson, R. H., Meyers, M. B., Spengler, B. A. and Biedler, J. L. (1983). Alteration of plasma membrane glycopeptides and gangliosides of Chinese hamster cells accompanying development of resistance to daunorubicin and vincristine. Cancer Res. 43, 222-228. Pilarski, L. M. and Belch, A. R. (1994). Circulating monoclonal B lineage cells expressing P-glycoprotein may be a reservoir of multidrug resistant disease in multiple myeloma. Blood 83, 724-736. Pileri, S. A., Sabattini, E., Falini, B., T&sari, P. L., Gherlinzoni, F., Michieli, M. G., Damiani, D., Zucchini, L., Gobbi, M., Tsuruo, T. and Baccarani, M. (1991). Immunohistochemical detection of the multidrug transport protein P170 in human normal tissues and malignant lymphomas. Histopathology 19, 131-140. Pinedo, II. M., Longo, D. L. and Chabner, B. A. (1992). Cancer Chemotherapy and Biological Response Modifiers. Annual 13. Elsevier, Amsterdam. Pirker, R., Wallner, J., Geissler, K., Linkesch, W., Haas, 0. A., Bettelheim, P., Scherrer, R., Valent, P., Havelec, L., Ludwig, H. and Lechner, K. (1991). MDRl gene expression and treatment outcome in acute myeloid leukemia. J. Natl. Cancer Inst. 83, 708-712.

C. Shustik et al.

70

Powis, S. J., Deverson, E. V., Coadwell, W. J., Ciruela, A., Huskisson, N. S., Smith, H., Butcher, G. W. and Howard, J. C. (1992). Effect of polymorphism of an MHC-linked transporter on the peptides assembled in a class I molecule. Nurure 357, 211-215. Presant, C. A., Kennedy, P. S., Wiseman, C., Gala, K., Bouzaglou, A. and Wyres, M. (1986). Verapamil reversal of clinical doxorubicin resistance in human cancer. A Wiltshire Oncology Medical Group pilot Phase I-II study. Am. J. Clin. Oncol. 9, 355-357.

Rao, V. V., Anthony, D. C. and Piwnica-Worms, D. (1994). MDRl monoclonal antibody C494 cross-reacts with pyruvate carboxylase.

gene-specific Cancer Res.

54, 1536-1541.

Raviv, Y., Pollard, H. B., Bruggemann, E. P., Pastan, I. and Gottesman, M. M. (1990). Photosensitized labeling of a functional multidrug transporter in living drug-resistant tumor cells. 1. Biol. Chem. 265, 3975-3980. Raymond, M., Rose, E., Housman, D. E. and Gros, P. (1989). Physical mapping, amplification, and over-expression of the mouse mdr gene family in multidrug resistance cells. Mol. Cell Biol. 10, 1642-1651. Raymond, M., Gros, P., Whiteway, M. and Thomas, D. Y. (1992). Functional complementation of yeast ste6 by a mammalian multidrug resistance gene. Science 256, 232-234.

Rich, D. P., Anderson, M. P., Gregory, R. J., Cheng, S. H., Paul, S., Jefferson, D. M., McCann, J. D., Klinger, K. W., Smith, A. E. and Welch, M. J. (1990). Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells. Nature 347, 358-363. Richert, N., Akiyama, S., Shen, D., Gottesman, M. M. and Pastan, I. (1985). Multiply drug resistant human KB carcinoma cells have decreased amounts of a 75 kDa and a 72 kDa glycoprotein. Proc. Natl. Acad. Sci. U.S.A. 82, 2330-2333. Riordan, J. R., Deuchars, K., Kartner, N., Alon, N., Trent, J. and Ling, V. (1985). Amplification of P-glycoprotein genes in tnultidrug resistant mammalian cell lines. Nature 316, 817-819. Riordan, J. R., Rommens, J. M., Kerem, B-S., Alow, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J. L., Drumm, M. L., Ianuzzi, M. C., Collins, F. S. and Tsui, L. C. (1989). Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245, 1066-1073.

Ro, J., Sahin, A., Ro, J. Y., Fritsche, H., Hortobagyi,

G. and Do, M. B. (1990). Immunohistochemical analysis of P-glycoprotein expression correlated with chemotherapy resistance in locally advanced breast cancer. Hum. Pathol. 21, 787-791.

P-Glycoprotein-Mediated

71

Multidrug Resistance

Roepe, P., Young, L., Cruz, J. and Carlson, D. (1993). Lower electrical membrane potential and altered pHi homeostasis multidrug-resistance (MDR) cells: further characterization of a serie of MDR cell lines expressing different levels of P-glycoprotein. Biochemistry 32, 11042-11056. Roepe, 1’. D. (1992). Analysis of the steady-state and initial rate of doxorubicin efflux from a series of multidrug-resistant cells expressing different levels of P-gl.ycoprotein. Biochemistry 31, 12555-12564. Roninson, I. B. (1983). Detection and mapping of homologous, repeated and amplified DNA sequences by DNA renaturation in-gel. Nucleic Acids Res 11, 5413-3423. I. B. (1991). Molecular and Cellular Biology of Multidrug Resistance in Tumor Cells. Plenum, New York.

Roninson,

Roninson, I. B., Abelson, H., Housman, D. E., Howell, N. and Varshavsky, A. (1984). Amplification of specific DNA sequences correlates with multidrug resistance in Chinese hamster cells. Nature 309, 626-628. Roninson, I. B., Chin, J. E., Choi, K., Gros, P., Housman, D. E., Fojo, A., Shen, D. W.:, Gottesman, M. M. and Pastan, I. (1986). Isolation of human MDRl DNA sequences amplified in multidrug resistant KB carcinoma cells. Proc. Natl. Acad. Sci. U.S.A.

83, 4538-4542.

Ross, D. D., Wooten, P. J., Sridhara, R., Ordonez, J. V., Lee, E. J. and Schiffer, C. A. (1993). Enhancement of daunorubicin accumulation, retention, and cytotoxicity by ver,apamil or cyclosporin A in blast cells from patients with previously untreated acute myeloid leukemia. Blood 82, 1288-1299. Rothenberg, M. L., Mickley, L. A., Cole, D. E., Balis, F. M., Tsuruo, T., Poplack, D. G. and Fojo, A. T. (1989). Expression of the MDRJIP-170 gene in patients witlh acute lymphoblastic leukemia. Blood 74, 1388-1395. Rubin S. C., Finstad, C. L., Hoskins, W. J., Saigo, P. E., Provencher, D. M., Federici, M. G., Hakes, T. B., Markman, M., Reichman, B. S., Lloyd, K. 0. and Lewis, J. L. 1(1990). Expression of P-glycoprotein in epithelial ovarian cancer: Evaluation as a marker of multidrug resistance. Am. J. Obstet. Gynecol. 69-73. Ruetz, S’. and Gros, P. (1994a). Functional expression of P-glycoprotein vesicles. J. Biol. Chem. 269, 12277-12284. Ruetz, S. and Gros, P. (1994b). Phosphatidylcholine for the mdr2 gene. Cell 77, 1071-1081.

translocase:

in secretory

A physiological role

Ruetz, S., Raymond, M. and Gros, P. (1993). Functional expression of P-glycoprotein encoded by the mouse mdr3 gene in yeast cells. Proc. Natl. Acud. Sci. U.S.A. 90, 11588-11592.

C. Shustik et al.

72

Safa, A. R. (1988). Photoaffinity labeling of the multidrug resistance related Pglycoprotein with photoactive analogs of verapamil. Proc. Nutl. Acad. Sci. U.S.A. 85, 7187-7191. Safa, A. R. (1993). Photoaffinity Cancer Invest. 11, 46-56.

labeling of P-glycoprotein

in multidrug resistant cells.

Safa, A. R., Glover, C. J., Meyers, M. B., Biedler, J. L. and Felsted, R. L. (1986). Vinblastine photoaffinity labeling of high molecular weight surface membrane glycoprotein specific for multidrug resistance cells. J. Biol. Chem. 261, 6137-6140. Safa, A. R., Glover, C. J., Sewell, J. L., Meyers, M. B., Biedler, J. L. and Felsted, R. L. (1987). Identification of the multidrug resistance-related membrane glycoprotein as an acceptor for calcium channel blockers. J. Biol. Chem. 262, 7884-7888.

Safa, A., Metha, N. D. and Agresti, M. (1989). Photoaffinity labelling of P-glycoprotein is multidrug resistant cells with photoactive analogs of colchicine. Biochem. Biophys. Rex Commun.

162, 1402-1408.

Safa, A. R., Agresti, M., Tamai, I., Mehta, N. D. and Vahabi, S. (1990a). The al adrenergic photoaffinity probe [12sI]arylazidoprazosin binds to a specific peptide of P-glycoprotein in multidrug resistant cells. Biochem. Biophys. Res. Commun. 166, 259-266.

Safa, A. R., Stern, R. K., Choi, K., Agresti, M., Tamai, I., Mehta, N. D. and Roninson, I. B. (1990b). Molecular basis of preferential resistance to colchicine in multidrug resistant human cells conferred by the G185V substitution in Pglycoprotein. Proc. Natl. Acad. Sci. U.S.A. 87, 7225-7229. Salmon, S. E., Grogan, T. M., Miller, T., Scheper, W. S. and Dalton, W. S. (1989). Prediction of doxorubicin resistance in vitro in myeloma, lymphoma, and breast cancer by P-glycoprotein staining. J. Natl. Cancer Inst. 81, 696-701. Salmon, S. E., Dalton, W. S., Grogan, T. M., Plezia, P., Lehnert, M., Roe, D. J. and Miller, T. P. (1991). Multidrug-resistant myeloma: laboratory and clinical effects of verapamil as a chemosensitizer. Blood 78, 44-50. Samuels, B. L., Mick, R., Vogelzang, N. J., Williams, S. F., Schilsky, R. L., Safa, A. R. and Ratain, M. J. (1993). Modulation of vinblastine resistance with cyclosporine: a Phase I study. Clin. Pharm. Ther. 54, 421429. Samuelson, J., Ayala, P., Oroczo, E. and Wirth, D. (1990). Emetine-resistant mutants of Entamoeba histolytica overexpress mRNAs for multidrug resistance. Mol. Biochem.

Parasitol. 38, 281-290.

Sanfilippo, 0.) Ronchi, E., De Marco, C., Di Fronzo, G. and Silvestrini, R. (1991). Expression of P-glycoprotein in breast cancer tissue and in vitro resistance to doxorubicin and vincristine. Eur. J. Cancer. 27, 155-158.

P-Glycoprotein-Mediated

Multidrug

Resistance

73

Sarkadi, YB., Price, E. M., Boucher, R. C., Germarm, U. and Scarborough, G. A. (1992). Expression of the human multidrug resistance cDNA in insect cells generates a high activity drug stimulated membrane ATPase. J. Biol. Chem. 267, 4854-4858.

Sato, H., Preisler, H., Day, R., Raza, A., Larson, R., Browman, G., Goldberg, J., Vogler, R., Grunwald, H., Gottlieb, A., Bennet, J., Gottesman, M. M. and Pastan, I. (1990). MDRl transcript levels as an indication of resistant disease in acute myelogenous leukemia. Br. J. Huematol. 75, 340-345. Schinkel, A. H., Roelofs, M. E. M. and Borst, P. (1991). Characterization of the human MDR3 P-glycoprotein and its recognition by P-glycoprotein specific monoclonal antibodies. Cancer Res. 51, 2628-2635. Schinkel, A. H., Smit, J. J. M., van Tellingen, O., Beijnen, J. H., Wagenaar, E., van Deemter, Mol., C. A. A. M., van der Valk, M. A., Robanus-Maandag, E. C., te Riele, H. P. J., Bems, A. J. M. and Borst, P. (1994). Disruption of the mouse m&la P-glycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs. Cell 77, 491-502. Schneider, J., Bak, M., Efferth, T., Kaufmann, M., Mattern, J. and Volm, M. (1989). P-glycoprotein expression in treated and untreated human breast cancer. BY. J. Cancer 60, 815-818.

of the Schurr, 13.) Raymond, M., Bell, J. and P. Gros. (1989). Characterization multidrug resistance protein expressed in cell clones stably transfected with the mouse mdrl cDNA. Cancer Res. 49, 2729-2734. Shapiro, A. B. and Ling, V. (1994). ATPase activity of purified and reconstituted P-glycoprotein from Chinese hamster ovary cells. J. Biof. Chem. 269, 3745-3754. Sharma, R. C., Inoue, S., Roitelman, J., Schimke, R. T. and Simoni, R. D. (19!)2). Peptide transport by the multidrug resistance pump. J. Biol. Chem. 267, 5731-5734.

Sharom, F. J., Yu, X. and Doige, C. A. (1993). Functional reconstitution of drug transport and ATPase activity in proteoliposomes containing partially purified P-glycoprotein. J. Biol. Chem. 268, 24197-24202. Sheppard, D. N., Rich, D. P., Ostedgaard, L. S., Gregory, R. J., Smith, A. E. and Welsh, M. J. (1993) Mutations in CFTR associated with mild-disease-form Cl- channels with altered pore properties. Nature 362, 160-164. Shimabuku, A. M., Nishimoto, T., Ueda, K. and Komano, T. (1992). P-glycoprotein: ATP hydrolysis by the N-terminal nucleotide-binding domain. J. Biol. Chem. 267, 4308-4311.

C. Shustik et al.

74

Shustik, C., Groulx, N. and Gros, P. (1991) Analysis of multidrug resistance (MDR-I) gene expression in chronic lymphocytic leukaemia (CLL). Br. J. Huematol. 79, 50-56. Skach, W. R (1994). Transmembrane orientation and topogenesis of the third and fourth membrane spanning regions of human P-glycoprotein (MDRI). Cancer Res. 54, 3202-3209. Skach, W. R., Calayag, M. C. and Lingappa, V. R. (1993). Evidence for alternate model of human P-glycoprotein structure and biogenesis. .I. Biol. Chem. 268, 6903-6908. Skovsgaard, T. (1978). Mechanism of cross-resistance between vincristine daunorubicin in Erlich ascites tumor cells. Cancer Res. 39, 4722-4727.

and

Slater, L. M., Sweet, P., Stupecky, M. and S. Gupta. (1986). Cyclosporin A reverses vincristine and daunorubicin resistance in acute lymphatic leukemia in vitro. J. Clin. Invest. 77, 1405-1408. Slovak, M. L., Hoeltege, G. A. and Ganapathi, R. (1986). Abnormally banded chromosomal regions in doxorubicin-resistant B16-BL6 murine melanoma cells. Cancer Res. 44, 4171-4177. Smit, J. J., Schinkel, A. H., OudeElferink, R. P., Groen, A. K., Wagenaar, E., Van Deemter, L., Mol, C. A., Ottenhofer, R., Van der Lugt, M. A., Van Roon, M. A., Van der Valk, M. A., Offerhaus, G. J., Berns, A. J. and Borst, P. (1993). Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease. Cell 75, 451-462. Solary, E., Caillot, D., Chauffert, B., Casasnovas, R. O., Dumas, M. and Maynadie, M. (1992). Feasibility of using quinine, a potential multidrug resistance-reversing agent, in combination with mitoxantrone and cytarabine for the treatment of acute leukemia. .I. Clin. Oncol. 10,1730-1736. Sonneveld, P., Durie, B. G., Lokhorst, H. M., Marie, J. P., Solbu, G., Suciu, S., Lowenberg, B. and Nooter, K. (1992a). Modulation of multidrug-resistant multiple myeloma by cyclosporin A. The Leukaemia Group of the EORTC and the HOVON. Luncet 340, 255-259. Sonneveld, P., Nooter, K., Burghouts, J. T. M., Herweijer, H., Adriaansen, H. J. and van Dongen, J. J. M. (1992b). High expression of the mdr3 multidrug-resistance gene in advanced-stage chronic lymphocytic leukemia. Blood 79, 1496-1500. Sonneveld, P., Durie, B. G. M., Lokhorst, H. M., Frutiger, Y., Schoester, M. and Vela, E. E. (1993). Analysis of multidrug-resistance (MDR-I) glycoprotein and CD56 expression to separate monoclonal gammopathy from multiple myeloma. Br. J. Huemutol.

83, 63-67.

P-Glycoprotein-Mediated

Multidrug

Resistance

75

Spies, T., Cerundolo, V., Colonna, M., Cresswell, P., Townsend, A. and DeMars, R. (1992). Presentation of viral antigen by MHC class I molecules is dependent on a putative peptide transporter heterodimer. Nature 355, 644-646. Sugimoto, Y. and Tsuruo, T. (1991). Development of multidrug resistance in rodent cell lines. In: Molecular and Cellular Biology of Multidrug Resistance in Tumor Cells (I. B. Roninson, ed.), pp. 3-55. Plenum, New York. Surin, B. P., Rosenberg, H. and Cox, G. B. (1985). Phosphate-specific of Escherichia coli: Nucleotide sequence and gene-polypeptide

transport system relationships. J.

Bacterial. 161, 189-198.

Tamai, I. and Safa, A. R. (1990). Competitive interaction of cyclosporins with the Vinca alkaloid binding site of P-glycoprotein in multidrug resistant cells. J. Biol. Chem. 265, 16509-16513. Tang Wai, D., Arnold, L., Brossi, A. and Gros, P. (1993). The nitrogen of the acetamido group of colchicine modulates P-glycoprotein mediated multidrug resistance. Biochemistry 32, 6470-6476. Tanigawara, Y., Okamura, N., Hirai, M., Yasuhara, M., Ueda, K., Kioka, N., Komano, T. and Hori, R. (1992). Transport of digoxin by human P-glycoprotein expressed in a porcine kidney epithelial cell line (LLC-PK,). J. Pharmacol. Exp. Ther. 263, 840-845.

Tatsuta, ‘I’., Naito, M., Oh-hara, T., Sugawara, I. and Tsuruo, T. (1992). Functional involvement of P-glycoprotein in blood-brain barrier. J. Biol. Chem. 267, 20383-20391.

Teeter, I,. D., Atsumi, S. I., Sen, S. and Kuo, T. (1986). DNA amplification in multidrug cross-resistant Chinese hamster ovary cells: Molecular charcaterization and cytogenetic localization of the amplified DNA. J. Cell Biol. 103, 1159-1166. Teeter, I,. D., Becker, F. F., Chisari, F. V., Li., D. and Kuo, M. T. (1990). Overexpression of the multidrug resistance gene mdr3 in spontaneous and chemically-induced mouse hepatocellular carcinomas. Mol. Cell Biol. 10,5728-5735. Thiebaut, F., Tsuruo, T., Hamada, H., Gottesman, M. M., Pastan, I. and Willingham. M. C. (1987). Cellular localization of the multidrug-resistance gene product P-gllycoprotein in normal tissues. Proc. Natl. Acad. Sci. U.S.A. 84, 7735-7738. Thiebaut, F., Tsuruo, T., Hamada, H, Gottesman, M. M., Pastan, I. and Willingham, M. C. (1989). Immunohistochemical localization in normal tissues of different epitopes in the multidrug transport protein P170: Evidence for localization in brain capillaries and cross-reactivity of one antibody with a muscle protein. J. Histochem.

Cytochem. 37, 159-164.

C. Shustik et al.

76

Thiebaut, F., Currier, S. J., Whitaker, J., Haugland, R. P., Gottesman, M. M., Pastan, I. and Willingham, M. C. (1990). Activity of the multidrug transporter results in alkalinization of the cytosol: Measurement of cytosolic pH by microinjection of a pH-sensitive dye. J. Histochem. Cytochem. 38, 685-690. Trezise, A. E. O., Romano, P. R., Gill, D. R., Hyde, S. C., Sepzlveda, F. V., Buchwald, M. and Higgins, C. F. (1992). The multidrug resistance and cystic fibrosis genes have complementary patterns of epithelial expression. EMBO J. 11, 4291-4303.

Trump, D. L., Smith, D. C., Ellis, P. G., Rogers, M. P., Schold, S. C., Winer, E. P., Jordan, V. C. and Fine, R. L. (1992). High-dose oral tamoxifen, a potentia1 multidrug-resistance-reversal agent: Phase I trial in combination with vinblastine. J. Natl. Cancer Inst. 84, 1811-1816.

Tsui, L. C. (1992). The spectrum

of cystic fibrosis mutations.

Trends

Genet.

8,

392-398.

Tsuruo, T., Iida, H., Tsukagoshi, S. and Sakurai, Y. (1981). Overcoming of vincristine resistance in P388 leukemia in vivo and in vitro through enhanced cytotoxicity of vincristine and vinblastine by verapamil. Cancer Res. 41, 1967-1972. Tsuruo, T., Iida, H., Tsukagoshi, S. and Sakurai, Y. (1982). Increased accumulation of vincristine and adriamycin in drug resistant tumor cells following incubation with calcium antagonists and calmodulin inhibitors. Cancer Res. 42, 4730-4733. Tsuruo, T., Iida, H., Ohkochi, E., Tsukagoshi, S. and Sakurai, Y. (1983). Establishment and properties of vincristine-resistant human myelogenous leukemia K562. Gann. 74, 751-758. Tsuruo, T., Iida, H., Kitatani, Y., Yokota, K., Tsukagoshi, S. and Sakurai, Y. (1984). Effects of quinidine and related compounds on cytotoxicity and cellular accumulation of vincristine and adriamycin in drug resistant tumor cells. Cancer Res. 44, 4303-4307.

Tsuruo, T., Iida, H., Yamashiro, M., Tsukagoshi, S. and Sakurai, Y. (1992b). Enhancement of vincristine- and adriamycin-induced cytotoxicity by verapamil in P388 leukemia and its sublines resistant to vincristine and adriamycin. Biochem. Pharmacol.

31, 3138-3140.

Ucci, G., Petrini, M., Riccardi, A., Invernizzi, R., Carulli, G., Luoni, R., Giordano, M. and Danova, M. (1992). Expression of ~170 protein in multiple myeloma: A clinical study. Zfematol. Oncoi. 10, 213-220. Ueda, K., Cardarelli, C., Gottesman, M. M. and Pastan, I. (1987). Expression of a full-length cDNA for the human MDRl gene confers resistance to colchicine, doxorubicin, and vinblastine. Proc. Natl. Acad. Sci. U.S.A. 84, 3004-3008.

P-Glycoprotein-Mediated

Multidrug

Resistance

77

Ueda, K., Okamura, N., Hirai, Y., Tanigawara, Y., Saeki, T., Kioka, N., Komano, T. and Hori, R. (1992). Human P-glycoprotein transports cortisol, aldosterone, and dexamethasone, but not progesterone. J. Biol. Chem. 267, 24248-24252. Valverde, M. A., Diaz, M., Sepulveda, F. V., Gill, D.’ R., Hyde, S. C. and Higgins, C. FT. (1992). Volume-regulated chloride channel associated with the human multidrug-resistance P-glycoprotein. Nature 355, 830-833. Van der :Bliek, A. M., Van der Velde-Koerts, T., Ling, V. and Borst, P. (1986). Overexpression and amplification of five genes in a multidrug resistant Chinese hamister ovary cell line. Mol. Cell. Biol. 6, 1671-1678. Van der Elliek, A. M., Kooiman, P. M., Schneider, C. and Borst, P. (1988). Sequence of &1DZ?3cDNA encoding a human P-glycoprotein. Gene 71, 401-411. Van der Valk, P., van Kalken, C. K., Ketelaars, H., Broxterman, H. J., Scheffer, G., Kuiper, C. M., Tsuruo, T., Lankelma, J., Meijer, C. J. and Pinedo, H. M. (1990). Distribution of multidrug resistance-associated P-glycoprotein in normal and neoplastic human tissues. Ann. Oncol. 1, 56-64. van der Zee, A. G. J., Hollema, H., de Jong, S., Boonstra, H., Goun, A., Willemse, P. H. B., Zijlstra, J. G. and de Vries, E. G. E. (1991). P-Glycoprotein expression and DNA topoisomerase I and II activity in benign tumors of the ovary and in malignant tumors of the ovary, before and after platinum/cyclophosphamide chemotherapy. Cancer Res. 51,5915-5920. Verrelle, P., Meissonier, F., Fonck, Y., Feillel, V., Dionet, C., Kwiatkowski, F., Plagne, R. and Chassagne, J. (1991). Clinical relevance of immunohistochemical detection of multidrug resistance P-glycoprotein in breast carcinoma. J. Nutl. Cancer Inst. 83, 111-116. Walker, J. E., Sraste, M., Runswick, M. J. and Gay, N. J. (1982). Distantly related sequences in the alpha-and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide-binding fold. EMBO J. 1, 945-951. Walworth, N. C. and Novick, P. J. (1987). Purification and characterization constitutive secretory vesicles from yeast. J. Cell Biol. 105,163-173.

of

Weinstein, R. S., Jakate, S. M., Dominguez, J. M., Lebovitz, M. D., Koukoulis, G. K., Klusens, L. F., Grogan, T. M., Saclarides, T. J., Roninson, I. B. and Coon, J. S. (1991). Relationship of the expression of the multidrug resistance gene product (P-glycoprotein) in human colon carcinoma to local tumor aggressiveness and lymph node metastasis. Cancer Res. 51, 2720-2726. Weinstein, R. S., Hansen, K. K., McBeath, R. B. and Dalton, W. S. (1993). Expression of the MDRl gene (P-glycoprotein) in breast cancer. Rec. Results Cancer Res. 127, 49-54.

78

C. Shustik

et al.

Wishart, G. C., Plumb, J. A., Going, J. J., McNicol, A. M., McArdle, C. S., Tsuruo, T. and Kaye, S. B (1990). P-glycoprotein expression in primary breast cancer detected by immunocytochemistry with monoclonal antibodies. Br. J. Cancer 62, 758-761. Wolf, D. C. and Horwitz, S. B. (1992). P-glycoprotein transports corticosterone photoaffinity-labeled by the steroid. Znt. J. Cancer 52, 141-146.

and is

Yahanda, A. M., Adler K. M., Fisher, G. A., Brophy, N. A., Halsey, J., Hardy, R. I, Gosland, M. P., Lum, B. L. and Sikic, B. I. (1993). Phase I trial of etoposide with cyclosporine as a modulator of multidrug resistance. J. Clin. Oncol. 10, 1624-1634. Yang, C. -P. H., Cohen, D., Greenberger, L. M., Hsu, S. I. -H. and Horwitz, S. B. (1990). Differential transport properties of two h4DR gene products are distinguished by progesterone. J. Biol. Chem. 265, 10281-10288. Yoshimura, A., Kuwazuru, Y., Sumizawa, T., Ichikawa, M., Ikeda, S. I., Ueda, T. and Akiyama, S. I. (1989). Cytoplasmic orientation and two domain structure of the multidrug transporter, P-glycoprotein, demonstrated with sequence specific antibodies. J. Biol. Chem. 264, 16282-16291. Yusa, K. and Tsuruo, T. (1989). Reversal mechanism of multidrug resistance by verapamil: direct binding of verapamil to P-glycoprotein on specific sites and transport of verapamil outward across the plasma membrane. Cancer Res. 49, 5002-5006. Zamora, J. M., Pearce, H. L. and Beck, W. T. (1988). Physico-chemical properties shared by compounds that modulate multidrug resistance in human leukemic cells. Mol. Pharmacol. 33, 454-462. Zhang, J. T. and Ling, V. (1991). Study of membrane orientation and glycosylated extracellular loops of mouse P-glycoprotein by in vitro translation. J. Biol. Chem. 266, 18224-18232. Zhang, J. T. and Ling, V. (1993). Membrane orientation of transmembrane segments 11 and 12 of MDR and non-MDR associated P-glycoproteins. Biochim. Biophys. Actu. 1153, 191-202. Zhang, J. T., Duthie, M. and Ling, V. (1993). Membrane topology of the N-terminal half of the hamster P-glycoprotein molecule. J. Biol. Chem. 268, 15101-15110.