Effect of the breast-cancer resistance protein on atypical multidrug resistance

Review

BCRP in atypical multidrug resistance

Effect of the breast-cancer resistance protein on atypical multidrug resistance Hermann Lage and Manfred Dietel

Simultaneous resistance of malignant cells to several antineoplastic agents that are structurally and functionally unrelated is known as multidrug resistance. It is one of the main causes of chemotherapy failure. Besides the classic multidrugresistant phenotype, mediated by increased activity of the ATP-binding cassette (ABC) transporter Pglycoprotein, there are other multidrug-resistant tumours, with resistance caused by different mechanisms. This is called atypical multidrug resistance. Pronounced overexpression of a novel ABC transporter has been observed in various human cancer cell lines with atypical multidrug resistance (which were established by in vitro exposure to mitoxantrone, topotecan, doxorubicin, or bisantrene). This novel transporter was originally named breastcancer resistance protein (BCRP). BCRP is a 655aminoacid protein of about 72 kDa. It can be thought of as an ABC ‘half-transporter’, and it forms dimers to produce an active transport complex. Transfection experiments with BCRP cDNA showed that the phenotype of atypical multidrug resistance could be transferred to formerly drug-sensitive cancer cells. Although the role of BCRP in drug resistance of clinical cancers is still unclear, preliminary data obtained by mRNA and protein expression analyses support the assumption that it has a role in clinical multidrug resistance. Lancet Oncology 2000; 1: 169–75

Since the introduction of chemotherapeutic strategies in the treatment of malignant diseases, oncologists have been confronted with the problem of resistance to therapy. Drug resistance can develop during administration of antineoplastic agents, as in small-cell lung carcinoma and acute lymphoblastic leukaemia, or it can be an inherent feature of a particular tumour type, such as non-small-cell lung carcinoma, pancreatic cancer and colorectal cancer. Moreover, aggravating circumstances are emerging: exposure of cancer cells to a single antitumour compound commonly results in cross-resistance to many other structurally and functionally unrelated chemotherapeutic agents to which the malignant cells have not been exposed. This process is known as pleiotropic or multidrug resistance (Figure 1). The original concept of multidrug resistance was introduced by Biedler and Riehm in 1970.1 By the mid-1970s a specific association had been found in cell culture models between a 170 kDa glycosylated cell-surface protein and a multidrug-resistant phenotype.2 Owing to the presumed THE LANCET Oncology Vol 1 November 2000

Figure 1. Confocal laser-scan microscopy image showing the atypical multidrug-resistant human gastric carcinoma cell line, EPG85-257RNOV, after exposure to fluorescent drugs. This treatment results in vesicular compartmentalisation of the antitumour agent and a drug-free nucleus.

involvement in drug permeation through hydrophobic membranes, the protein was designated P-glycoprotein (P for permeability). This protein was purified in 1979,3 and strong evidence of its role in multidrug resistance came in 1982, when Debenham and colleagues showed that transfer of DNA from multidrug-resistant to non-resistant cells conferred a multidrug-resistant phenotype in association with expression of P-glycoprotein.4 The P-glycoproteinencoding MDR-1 gene was cloned in 1985,5 and its function as an energy-dependent pump was postulated on the basis of sequence homologies with a prokaryotic haemolysin transport protein.6 Meanwhile, it became clear that P-glycoprotein is a member of the ATP-binding cassette transmembrane transporter superfamily (ABC transporters), the largest protein family known to date. This superfamily includes hundreds of polypeptides derived from all living cells, from bacteria to mammals.7 Most of the known members of the ABC-transporter family are active membrane-embedded pumps; that is, they transport their substrates against a concentration gradient by means of energy from ATP hydrolysis. The essential structural requirements for an active ABC transporter seem to be two membraneanchoring transmembrane domains, which generally consist of six transmembrane helices, and two nucleotide-binding HL and MD are at the Institute of Pathology, University Hospital Charité, Charité Campus Mitte, Humboldt-University Berlin, Schumannstrasse 20/21, D-10117 Berlin, Germany. Tel: +49 30 2802 2261. Fax: +49 30 2802 3407. E-mail: [email protected]

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BCRP in atypical multidrug resistance

regions necessary for ATP hydrolysis. These functional domains can be built by one polypeptide chain, or can be a multiprotein complex. The full-length ABC transporter P-glycoprotein (or ABCB1, according to the recommendations of the Human Gene Nomenclature Committee) is located in the cytoplasmic membrane, on the apical or luminal surface of polarised epithelial cells. This transporter can extrude a wide range of structurally unrelated lipophilic xenobiotics, including clinically important antitumour agents, such as anthracyclines, mitoxantrone, taxanes, epipodophyllotoxins, and vinca alkaloids. P-glycoprotein transports these antineoplastic drugs in unmodified form. Precise analysis of the molecular mechanisms resulting in a multidrug-resistant phenotype, such as the investigation of P-glycoprotein, is of major clinical interest, because full understanding of these mechanisms will provide the basis for the development of strategies to circumvent multidrug resistance. These strategies include the application of multidrug-resistance-modulating compounds, or chemosensitisers, such as the ciclosporin A analogue PSC 833, a non-competitive inhibitor of P-glycoprotein. However, clinical trials with multidrug-resistance modulators that inhibit P-glycoprotein showed little clinical success, probably because in the clinical situation several mechanisms are simultaneously or successively active. Other multidrug-resistant cell lines showed no Pglycoprotein expression, but they did show cross-resistance similar to that caused by P-glycoprotein. Thus, the multidrug-resistant phenotype mediated by P-glycoprotein is now designated the classical form, whereas a multidrugresistant phenotype in the absence of P-glycoprotein is designated as atypical multidrug resistance. Various mechanisms have been associated with atypical multidrug resistance; for example, alterations in drug targets such as DNA topoisomerase II;8 vesicular compartmentalisation of antitumour agents;9 increased detoxification of compounds by glutathione-S-transferases;10 overexpression of the lungresistance-related protein (the major vault protein in the transporter core of the nuclear core complex11); changes in DNA-repair mechanisms;12 modulation of factors involved in the regulation of apoptosis;13 and increased activity of alternative ABC transporters. Thus, multidrug resistance, once thought to be completely explicable by increased expression of P-glycoprotein, is far more complex and involves many more factors. An example of an atypical multidrug-resistant, Pglycoprotein negative cell line is shown in Figure 1. The human gastric carcinoma cell line, EPG85-257RNOV, was established by in vitro selection with the anthracenedione mitoxantrone. Exposure of these drug-resistant cells to antineoplastic drugs, such as mitoxantrone or daunorubicin, results in vesicular compartmentalisation of the antitumour agent and a drug-free nucleus.9 The molecular mechanism for this compartmentalisation of drugs is still unclear. However, a possible mechanism is the energy-dependent transport of the drugs into the vesicles by an alternative ABC-transporter. A contribution to antineoplastic-drug resistance has been demonstrated for some, but not all, of the known ABC transporters. The multidrug resistance protein (MRP),14 also 170

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Figure 2. Comparison of band patterns in various human multidrugresistance cell lines from our laboratory, using the mRNA differential display approach.28 In each case, samples prepared from two independent cell passages were used. In the human gastric carcinoma cell line EPG85-257RNOV there are increased amounts of two BCRPrepresenting PCR products of different size (indicated by arrows). The identity of both amplification products with the BCRP-encoding sequence was confirmed after excision from the gel, reamplification by PCR, cloning into a plasmid vector, clonal propagation in Escherichia coli, and terminal sequencing. These PCR fragments would be useful for cloning a full-length cDNA encoding the complete BCRP gene.

known as MRP1 or ABCC1, can transport antitumour agents either conjugated to anionic ligands such as glutathione, glucoronide, or sulphate, or in unmodified form, possibly together with glutathione. The canalicular multispecific organic anion transporter (cMOAT),15 also known as MRP2 or ABCC2, confers resistance to cisplatin, etoposide, methotrexate, and vincristine. MRP316 (synonyms MOAT-D and ABCC3) confers resistance to etoposide, methotrexate, and vincristine. Resistance to thiopurine anticancer drugs, such as 6-mercaptopurine and thioguanine, is conferred by MRP517 (MOAT-C or ABCC5). A slight increase in drug tolerance to etoposide was shown in transfection experiments with cDNA for rat TAP1 (ABCB2) and TAP2 (ABCB3), encoding the subunits of the heterodimeric ‘transporter associated with antigen THE LANCET Oncology Vol 1 November 2000

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Review

BCRP in atypical multidrug resistance

For illustration of convenient cloning strategies, Figure 2 shows a differential display gel, comparing various atypical multidrug-resistant cell lines. Specific BCRP signals were detected in the cell line EPG85-257RNOV, derived from human gastric carcinoma, which shows a mitoxantrone-induced atypical multidrugresistance phenotype.9

Transmembrane domain

TM6

TM5

TM4

TM3

TM2

TM1

Outside

Cytosol COOH

Characteristics

According to the recommendations of the Human Gene Nomenclature Committee, the atypical-multidrugresistance-associated ABC transNucleotide-binding porter indicated with the synonyms domain BCRP, MXR, and ABCP is designated ABCG2. To prevent confusion, NH2 throughout this review we use the first published term for this transport Figure 3. Hypothetical protein structure of the human ABC ‘half-transporter’ BCRP and related molecule – BCRP. transport molecules. The active ABC-transport complex consists of two half-transporters. It is The human BCRP gene was proposed that the transmembrane domains form a channel-like structure serving to translocate substrates. TM = transmembrane helix. mapped to chromosome 4q22.31 In various drug-resistant cell lines, processing’ (TAP).18 So far there is no evidence that the other transcription of the gene gives a 2.4 kb mRNA encoding a members of the MRP or ABCC subfamily of ABC putative 655-aminoacid, 72.6 kDa polypeptide, which transporters (MRP4 [MOAT-B or ABCC4] and MRP6 seems to be predominantly localised in the plasma [ABCC6]), are involved in resistance to common anticancer membrane rather than in internal membranes.32–34 The mouse homologous specific cDNA, Bcrp1,35 encodes a 657drugs. In contrast to these observations, several studies have aminoacid polypeptide chain corresponding closely in shown that in vitro selection with the anthracenedione sequence and structure to human BCRP. The sequences are mitoxantrone results in an atypical multidrug-resistant 81% identical and 86% homologous. Furthermore, BCRP phenotype characterised by reduced intracellular drug has 31% identity with the white gene of Drosophila accumulation, not caused by overexpression of any of the melanogaster and 30% identity with the human homologue ABC transporters mentioned above.9,19–27 Thus, a novel of that gene, ABC8, also designated as White or ABCG1. alternative drug-transport mechanism is mediating the Both BCRP and ABC8 belong to one of the seven human atypical multidrug resistance in these cell lines. Different subfamilies of ABC transport proteins, ASBCG. The experimental strategies were used by several investigators to homologous White subfamily in D melanogaster consists of clone and characterise this putative new transport molecule. the white, scarlet, and brown genes. These genes encode ABC-transporter subunits, necessary for the transport of A novel ABC transporter in cells with atypical pigment precursors into cells responsible for eye colour.36 However, the physiological role of the human ABC8 gene is multidrug resistance Using an RNA fingerprinting approach, a modification of not yet known. P-glycoprotein and the MRP subfamily the mRNA differential display technique,28 this transport members represent full transporter molecules. By contrast, molecule was identified as ‘breast-cancer resistance protein’ the members of the ABCG and White subfamilies are about (BCRP)29 in the anthracycline-selected human breast-cancer half the size of full-length ABC-transporter proteins. Their cell line MCF-7/AdrVp. An alternative cDNA encoding the domain arrangement is characterised by an N-terminal novel ABC transporter was cloned from the mitoxantrone- single hydrophilic ATP-binding cassette or nucleotideselected human colon-carcinoma cell line S1-M1-80, by a binding region and a hydrophobic transmembrane domain, differential hybridisation screening strategy, and therefore probably consisting of six transmembrane helices, near the designated ‘mitoxantrone-resistance associated protein’ C-terminal end (Figure 3). In D melanogaster, the half(MXR).30 Simultaneously, a third approach, based on transporter polypeptides encoded by the white, scarlet, and searching of the express sequence tag database for gene brown genes form active transporter molecules after products of ABC-transporter-encoding sequences, led to heterodimerisation.36 identification of the same transcript.31 Because of high mRNA expression in the placenta, that gene product was Subunits required for an active transmembrane designated ‘placenta-specific ABC gene’ (ABCP). However, transporter this novel polypeptide seems likely to be the long-sought Because the BCRP polypeptide chain has the characteristics transporter leading to an atypical multidrug-resistance of a half-transporter, consisting of one nucleotide-binding phenotype characterised by high resistance to mitoxantrone. region and one transmembrane domain (Figure 3), an THE LANCET Oncology Vol 1 November 2000

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BCRP in atypical multidrug resistance

MX MX

MX

Outside

Review

MX MX

MX

Expression of BCRP in multidrug-resistant cell lines and normal human tissues

Various different cell lines with atypical multidrug resistance strikingly overexpress BCRP or Bcrp1 MX ATP ADP+Pi specific mRNA.35,40 Sources of these cell lines include carcinomas of Vesicular MX breast, colon, stomach, and ovary compartments MX as well as fibrosarcoma and MX myeloma. Furthermore, a multidrugMX ATP ADP+Pi Cytosol resistant phenotype can be MX transferred to non-resistant cells MX by transfection with BCRP cDNA.29 MX MX Induction of BCRP overexpression results from continuous in vitro MX exposure of tumour cells with Endoplasmic reticulum MX ATP ADP+Pi mitoxantrone, doxorubicin, bisantrene, or topotecan. In some of these cell lines, there is MX MX Nucleus pronounced amplification of the chromosomal regions corresponding to the BCRP or Bcrp1 genes.35,39,40 Northern-blot and dot-blot Figure 4. Various possibilities of action of BCRP. Immunocytological analyses show that BCRP is hybridisation experiments31 have predominantly located in the cytoplasmic membrane. In an ATP-dependent manner, drugs could be shown that BCRP mRNA expression extruded from the cell by BCRP. BCRP pump activity could also contribute to vesicular is highest in the placenta, but it is also compartmentation of cytotoxic drugs or facilitate phase II drug metabolism by carrying xenobiotic seen in heart, ovary, kidney, and fetal substances into the lumen of the endoplasmic reticulum. MX, mitoxantrone. liver. Another study found weak intensive search is underway for other transporter BCRP mRNA expression in liver, colon, small intestine, subunits that may participate in the BCRP transmembrane testis, prostate, and brain.29 The discrepancies between the transport complex. Transfection experiments showed that studies, for heart and kidney, may be the result of different BCRP-transfected clones became drug-resistant, had raw material and the problems of detecting mRNA that diminished intracellular daunorubicin accumulation, and occurs in low amounts by hybridisation techniques. The had an ATP-dependent increase in rhodamine-123 use of monoclonal antibodies against BCRP will help to efflux.29,32,37 However, all these experiments have not elucidate its distribution in normal human tissues. answered the question of which subunits are required for BCRP action. Substrates for BCRP In analogy to the transport complexes formed by The established drug-resistant cancer cell lines expressing the members of the D melanogaster White subfamily BCRP and Bcrp1 mRNA show similar but not identical and human ABC-half-transporters such as TAP cross-resistance patterns (Tables 1 and 2). In common, (a heterodimer of TAP1 and TAP2), the activity of BCRP the cells show characteristically high cross-resistance to is very likely to require dimerisation. However, potential the anthracenedione mitoxantrone, anthracyclines such as molecules for heterodimerisation with BCRP are not daunorubicin and doxorubicin, topotecan, bisantrene, and yet known. Ross38 has suggested that tumour cells may SN-38, the active form of irinotecan. Moreover, the use versatile transporter subunit combinations to BCRP-mediated multidrug resistance is characterised by customise transport for different anticancer drugs. Under retained sensitivity to cisplatin, paclitaxel, and vinca that hypothesis, three different hypothetical transport alkaloids such as vincristine and vinblastine. Discrepant complex subunits could generate up to six functional cross-resistance patterns between cell lines may be caused dimeric structures, if both homodimers and heterodimers by the simultaneous activity of other transport proteins could be formed. A combination of four hypothetical such as MRP, other drug-resistance mechanisms, or subunits could generate up to ten different protein alternative dimerisation of the full BCRP-containing dimers. However, cytogenetic analyses of breast and complex. Thus, typical substrates of the classical multidrugcolon carcinoma cell lines (MCF-7/MX, MCF- resistance mediator P-glycoprotein, such as vinca alkaloids, 7/AdVp3000, S1-M1-80) that overexpress BCRP mRNA paclitaxel, and verapamil, are not transported by and show amplification of BCRP-encoding chromosomal BCRP. Thus, the atypical multidrug-resistance phenotype regions of chromosome 4 found no coamplification of caused by BCRP overlaps with, but is distinct from, other chromosomal regions.39 A possible explanation of the classical multidrug-resistance due to P-glycoprotein this observation is that a potential partner molecule activity. Moreover, BCRP activity seems to confer of BCRP may not be overexpressed, and instead higher resistance to mitoxantrone and topotecan than BCRP homodimerises to form an active transporter. P-glycoprotein does. MX

?

?

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Review

BCRP in atypical multidrug resistance

Table 1. Cross-resistance pattern in human cancer cell lines expressing BCRP Cell line

Selecting agent

Cross-resistance to

Sensitive to

Reference

None Mitoxantrone Mitoxantrone Doxorubicin Doxorubicin Topotecan Mitoxantrone

DB, DOX, MX, SN-38, TPT CIS, DOX, ETO, VBL, VCR SN-38, TPT DB, MX, TPT BIS, DB, MX, TAX, TPT, VBL MX, SN-38, CAM, DOX, ETO, TAX

CIS, TAX, VCR

29, 38 22 23 21, 26, 33 26, 32 47 Not published†

Mitoxantrone Mitoxantrone Mitoxantrone

DOX, TPT BIS, DB, EPI, SN-38, TPT DB, DOX

TAX, VBL CIS, TAX, VCR

30, 37 26, 32, 41 48

Mitoxantrone

CAM, DB, DOX, SN-38, TAX, TPT

CIS, VBL, VCR

9, 25, 49

Mitoxantrone

DOX, TAX

CIS, DB, VCR

48

Breast carcinoma MCF-7/BCRP clone8* MCF-7/Mitox MCF-7/MX MCF-7/AdrVp1000 MCF-7/AdVp3000 MCF-7/TPT300 MDA-MB-231RNOV

CIS, TAX, VCR

Colon carcinoma S1M1-3.2 S1-M1-80 HT29RNOV Gastric carcinoma EPG85-257RNOV Fibrosarcoma EPF086-079RNOV Myeloma 8226/MR20

Mitoxantrone

50

Ovarian carcinoma Igrov1 T8 Igrov1 MX3

* BCRP transfected cell line.

Topotecan Mitoxantrone

CAM, MX, SN-38 SN-38, TPT

CIS, DOX, TAX CAM, CIS, DOX, TAX

51 51

† Established in our laboratory.

BIS, bisantrene; CAM, camptothecin; CIS, cisplatin; DB, daunorubicin; DOX, doxorubicin; ETO, etoposide; EPI, epirubicin; MX, mitoxantrone; SN-38, 7-ethyl-10-hydroxycamptothecin; TAX, paclitaxel; TPT, topotecan; VBL, vinblastine; VCR, vincristine.

Moreover, BCRP could facilitate phase II drug Experimental data have given no evidence for the normal metabolism by transport of anticancer compounds into the role and the physiological substrates of BCRP, so there is lumen of the endoplasmic reticulum, which is where wide scope for speculation. First, the very high BCRP conjugation of various substances with glucuronic acid expression in normal placenta suggests that this transport takes place. BCRP-expressing cells are capable of molecule may have a role in maintaining the placental glucuronidating antineoplastic agents.41 barrier. In that case, BCRP could be involved in the translocation of essential, not yet identified, substances Reversal of BCRP-dependent atypical multidrug from maternal to fetal circulation. However, because BCRP resistance by pharmacological agents was identified in the context of multidrug resistance, it is Successful reversal of a multidrug-resistant phenotype more likely to be involved in protecting the fetus from would be the basis of an effective drug-based therapeutic strategy. Unfortunately, drugs that modulate classical harmful substances. Although immunocytological findings show that multidrug resistance, such as P-glycoprotein inhibitors BCRP, in contrast to all known ABC half-transporters, verapamil, calmodulin antagonists, and ciclosporins, is mainly present at the plasma membrane in cancer cells32–34 do not affect BCRP-mediated atypical multidrug and is, therefore, likely to be involved in active resistance. Nevertheless, a novel and specific inhibitor drug transport from the cell, we cannot exclude the of BCRP activity has been identified, which does possibility that it is also present on internal cellular not reverse multidrug resistance in cells that overexpress membranes of the endoplasmatic reticulum or vesicular P-glycoprotein or MRP.37,42 This compound, isolated structuresin tumour cells and in normal tissue (Figure 4). from Aspergillus fumigatis, is known as fumitremorgin In that case, BCRP could be involved in the formation of C. The mechanism of interaction of fumitremorgin C cytoplasmic mitoxantrone-containing vesicles, which have with BCRP is unknown, but it is likely to interact been observed in the BCRPoverexpressing gastric carcinoma cell Table 2. Cross-resistence pattern in murine fibroblast cell lines expressing Bcrp1 line EPG85-257RNOV9 (Figure 1) Selecting agent Cross-resistance to Sensitive to Reference and in various sublines of the Cell line colon carcinoma cell line S1.32 Under Fibroblast cells Mitroxantrone BIS, DOX, ETO, TPT CIS, DB, TAX, VCR 35 physiological conditions, this M32 Topotecan BIS, DOX, ETO, MX CIS, DB, TAX, VCR 35 mechanism could be involved T6400 Doxorubicin BIS, DB, ETO, MX, TPT CIS, TAX, VCR 35 in cellular translocation and D320 BIS, bisantrene; DB, daunorubican; DOX, doxorubicin; ETO, etoposide; MX, mitoxantrone; TAX, paclitaxel; distribution procedures of not yet TPT, topotecan; VCR, vincristene. characterised metabolites.

Putative physiological role of BCRP

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BCRP in atypical multidrug resistance

Search strategy and selection criteria Published and unpublished data for this review were identified by searches of the PubMed database provided by the National Center for Biotechnology Information at the National Institutes of Health, Bethesda, MD, USA, and references from relevant articles. We also contacted researchers to obtain preliminary data. directly with the ABC-transport molecule because it consists of a planar, multi-ring structure like mitoxantrone and the anthracyclines and therefore may compete with these agents for the binding sites on BCRP. Whether fumitremorgin C is suitable for clinical use has to be proven in further studies. Another potential BCRP-inhibiting, but not specific, compound is the acridonecarboxamide derivative GF120918, a secondgeneration P-glycoprotein antagonist.43 It was able to resensitise BCRP-expressing cells against treatment with mitoxantrone.44

Clinical relevance of BCRP Investigation of how frequently BCRP occurs in human tumours and its importance as a prognostic indicator is at a very early stage. Experimentation in this area will be facilitated by the availability of tumour specimens and reagents for the sensitive and specific detection of BCRP mRNA and polypeptide. In one study, with measurement of BCRP mRNA expression by a semiquantitative reverse transcriptase PCR technique, expression was detected in blast cells from 21 patients with acute leukaemia (20 acute myeloid leukaemia, one acute lymphocytic leukaemia).45 High expression was detected in seven of these patients (33%), whose leukaemia was drug resistant, whereas the remaining patients showed low or barely detectable expression. A similar PCR-based approach on samples from patients with ovarian carcinoma showed high BCRP expression in 17 of 58 cases (29%).46 Substantial improvement in detection of BCRP in routinely obtained tumour samples will come with the development of antibodies against the polypeptide chain. Meanwhile, recent developments include a polyclonal BCRP-specific antiserum, anti-MXR 87405,32,33 suitable for western blotting and immunohistochemistry, and the tested in monoclonal antibody BXP-3434 immunohistochemistry. Use of BXP-34 on cryosections of a panel of primary and drug-treated human tumour samples revealed weak BCRP-specific staining in one small-intestinal adenocarcinoma.34 In all other tumour samples (n=56), including those from patients with breast carcinoma and acute myeloid leukaemia, previously exposed to chemotherapy or not, no detectable BCRP polypeptide was observed. However, these preliminary data obtained by mRNA expression analysis and protein expression analysis in various human cancers are not consistent.

Conclusion The characterisation of mechanisms causing atypical multidrug resistance is of fundamental importance for the 174

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development of strategies to prevent or circumvent resistance of treatment. In vitro, tumour cells with atypical multidrug resistance defend themselves against cytotoxic attack by switching on the novel ABC-transporter BCRP, which seems to be the long-sought mitoxantrone-resistance factor. Inhibition of its action is likely to give significant therapeutic benefit. Whether BCRP expression is raised in human cancers is still not clear. However, so that we can discover the effect of BCRP on clinical multidrug resistance, we need more extended studies with improved techniques suited to large-scale BCRP screening of human tumour specimens. Acknowledgments

Our contributions about atypical multidrug resistance cited in this paper were supported by grants from NOVARTIS-Stiftung für therapeutische Forschung and Deutsche Krebshilfe (grant number 101313-La 3). We thank Klaus Mantwill for technical assistance. References

1 Biedler JL, Riehm H. Cellular resistance to actinomycin D in Chinese hamster cells in vitro: cross-resistance, radioautographic, and cytogenetic studies. Cancer Res 1970; 30: 1174–84. 2 Juliano RL, Ling V. A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim Biophys Acta 1976; 455: 152–62. 3 Riordan JR, Ling V. Purification of P-glycoprotein from plasma membrane vesicles of Chinese hamster ovary cell mutants with reduced colchicine permeability. J Biol Chem 1979; 254: 12701–05. 4 Debenham PG, Kartner N, Siminovitch L, Riordan JR, Ling V. DNA-mediated transfer of multiple drug resistance and plasma membrane glycoprotein expression. Mol Cell Biol 1982; 2: 881–89. 5 Riordan JR, Deuchars K, Kartner N, Alon N, Trent J, Ling V. Amplification of P-glycoprotein genes in multidrug-resistant mammalian cell lines. Nature 1985; 316: 817–19. 6 Chen CJ, Chin JE, Ueda K, Clark DP, Pastan I, Gottesman MM, Roninson IB. Internal duplication and homology with bacterial transport proteins in the mdr1 (P-glycoprotein) gene from multidrug-resistant human cells. Cell 1986; 47: 381–89. 7 Higgins CF. ABC transporters: from microorganisms to man. Annu Rev Cell Biol 1992; 8: 67–113. 8 Beck WT, Cirtain MC, Danks MK, et al. Pharmacological, molecular, and cytogenetic analysis of ‘atypical’ multidrug-resistant human leukemic cells. Cancer Res 1987; 47: 5455–60. 9 Dietel M, Arps H, Lage H, Niendorf A. Membrane vesicle formation due to acquired mitoxantrone resistance in human gastric carcinoma cell line EPG85-257. Cancer Res 1990; 50: 6100–06. 10 Peters WHM, Roelofs HMJ. Biochemical characterization of resistance to mitoxantrone and adriamycin in Caco-2 human adenocarcinoma cells: a possible role for glutathione S-transferases. Cancer Res 1992; 52: 1886–90. 11 Scheper RJ, Broxterman HJ, Scheffer GL, et al. Overexpression of a M(r) 110,000 vesicular protein in non-P-glycoprotein-mediated multidrug resistance. Cancer Res 1993; 53: 1475–79. 12 Deffie AM, Alam T, Seneviratne C, et al. Multifactorial resistance to adriamycin: relationship of DNA repair, glutathione transferase activity, drug efflux, and P-glycoprotein in cloned cell lines of adriamycin-sensitive and -resistant P388 leukemia. Cancer Res 1988; 48: 3595–602. 13 Desoize B Anticancer drug resistance and inhibition of apoptosis. Anticancer Res 1994; 14: 2291–94. 14 Cole SP, Bhardwaj G, Gerlach JH, et al. Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line. Science 1992; 258: 1650–54. 15 Taniguchi K, Wada M, Kohno K, et al. A human canalicular multispecific organic anion transporter (cMOAT) gene is overexpressed in cisplatin-resistant human cancer cell lines with decreased drug accumulation. Cancer Res 1996; 56: 4124–29. 16 Kool M, van der Linden M, de Haas M,et al. MRP3, an organic anion transporter able to transport anti-cancer drugs. Proc Natl Acad Sci USA 1999; 96: 6914–19. 17 Wijnholds J, Mol CA, van Deemter L, et al. Multidrug-resistance protein 5 is a multispecific organic anion transporter able to transport nucleotide analogs. Proc Natl Acad Sci USA 2000; 97: 7476–81. THE LANCET Oncology Vol 1 November 2000

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18 Izquierdo MA, Neefjes JJ, Mathari AE, Flens MJ, Scheffer GL, Scheper RJ. Overexpression of the ABC transporter TAP in multidrug-resistant human. Br J Cancer 1996; 74: 1961–67. 19 Dalton WS, Cress AE, Alberts DS, Trent JM. Cytogenetic and phenotypic analysis of a human colon carcinoma cell line resistant to mitoxantrone. Cancer Res 1988; 48: 1882–88. 20 Harker WG, Slade DL, Dalton WS, Meltzer PS, Trent JM. Multidrug resistance in mitoxantrone-selected HL-60 leukemia cells in the absence of P-glycoprotein overexpression. Cancer Res 1989; 49: 4542–49. 21 Chen YN, Mickley LA, Schwartz AM, Acton EM, Hwang JL, Fojo AT. Characterization of adriamycin-resistant human breast cancer cells which display overexpression of a novel resistance-related membrane protein. J Biol Chem 1990; 265: 10073–80. 22 Taylor CW, Dalton WS, Parrish PR, et al. Different mechanisms of decreased drug accumulation in doxorubicin and mitoxantrone resistant variants of the MCF7 human breast cancer cell line. Br J Cancer 1991; 63: 923–29. 23 Nakagawa M, Schneider E, Dixon KH, et al. Reduced intracellular drug accumulation in the absence of P-glycoprotein (mdr1) overexpression in mitoxantrone-resistant human MCF-7 breast cancer cells. Cancer Res 1992; 52: 6175–81. 24 Yang CJ, Horton JK, Cowan KH, Schneider E. Cross-resistance to camptothecin analogues in a mitoxantrone-resistant human breast carcinoma cell line is not due to DNA topoisomerase I alterations. Cancer Res 1995; 55: 4004–09. 25 Kellner U, Hutchinson L, Seidel A, et al. Decreased drug accumulation in a mitoxantrone-resistant gastric carcinoma cell line in the absence of P-glycoprotein. Int J Cancer 1997; 71: 817–24. 26 Lee JS, Scala S, Matsumoto Y, et al. Reduced drug accumulation and multidrug resistance in human breast cancer cells without associated P-glycoprotein or MRP overexpression. J Cell Biochem 1997; 65: 513–26. 27 Hazlehurst LA, Foley NE, Gleason-Guzman MC, et al. Multiple mechanisms confer drug resistance to mitoxantrone in the human8226 myeloma cell line. Cancer Res 1999; 59: 1021–28. 28 Liang P, Pardee AB. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 1992; 257: 967–71. 29 Doyle LA, Yang W, Abruzzo LV, et al. A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc Natl Acad Sci USA 1998; 95: 15665–70. 30 Miyake K, Mickley L, Litman T, et al. Molecular cloning of cDNAs which are highly overexpressed in mitoxantrone-resistant cells: demonstration of homology to ABC transport genes. Cancer Res 1999; 59: 8–13. 31 Allikmets R, Schriml LM, Hutchinson A, Romano-Spica V, Dean M. A human placenta-specific ATP-binding cassette gene (ABCP) on chromosome 4q22 that is involved in multidrug resistance. Cancer Res 1998; 58: 5337–39. 32 Litman T, Brangi M, Hudson E, et al. The multidrug-resistant phenotype associated with overexpression of the new ABC halftransporter, MXR (ABCG2). J Cell Sci 2000; 113: 2011–21. 33 Rocchi E, Khodjakov A, Volk EL, et al. The product of the ABC half-transporter gene ABCG2(BCRP/MXR/ABCP) is expressed in the plasma membrane. Biochem Biophys Res Commun 2000; 271: 42–46. 34 Scheffer GL, Maliepaard M, Pijnenborg AC, et al. Breast cancer resistance protein is localized at the plasma membrane in mitoxantrone- and topotecan-resistant cell lines. Cancer Res 2000; 60: 2589–93.

THE LANCET Oncology Vol 1 November 2000

BCRP in atypical multidrug resistance

35 Allen JD, Brinkhuis RF, Wijnholds J, Schinkel AH. The mouse Bcrp1/Mxr/Abcp gene: amplification and overexpression in cell lines selected for resistance to topotecan, mitoxantrone, or doxorubicin. Cancer Res 1999; 59: 4237–41. 36 Griffin J, Sansom C. The transporter fact book. San Diego: Academic Press, Harcourt Brace & Company, 1998: 115–20. 37 Rabindran SK, Ross DD, Doyle LA, Yang W, Greenberger LM. Fumitremorgin C reverses multidrug resistance in cells transfected with the breast cancer resistance protein. Cancer Res 2000; 60: 47–50. 38 Ross DD. Novel mechanisms of drug resistance in leukemia. Leukemia 2000; 14: 467–73. 39 Knutsen T, Rao VK, Ried T, et al. Amplification of 4q21-q22 and the MXR gene in independently derived mitoxantrone-resistant cell lines. Genes Chromosomes Cancer 2000; 27: 110–16. 40 Ross DD, Yang W, Abruzzo LV, et al. Atypical multidrug resistance: breast cancer resistance protein messenger RNA expression in mitoxantrone-selected cell lines. J Natl Cancer Inst 1999; 91: 429–33. 41 Brangi M, Litman T, Ciotti M, et al. Camptothecin resistance: role of the ATP-binding cassette (ABC), mitoxantrone-resistance halftransporter (MXR), and potential for glucuronidation in MXRexpressing cells. Cancer Res 1999; 59: 5938–46. 42 Rabindran SK, He H, Singh M, et al. Reversal of a novel multidrug resistance mechanism in human colon carcinoma cells by fumitremorgin C. Cancer Res 1998; 58: 5850–58. 43 Hyafil F, Vergely C, Du Vignaud P, Grand-Perret T. In vitro and in vivo reversal of multidrug resistance by GF120918, an acridonecarboxamide derivative. Cancer Res 1993; 53: 4595–602. 44 de Bruin M, Miyake K, Litman T, Robey R, Bates SE. Reversal of resistance by GF120918 in cell lines expressing the ABC halftransporter, MXR. Cancer Lett 1999; 146: 117–26. 45 Ross DD, Karp JE, Chen TT, Doyle LA. Expression of breast cancer resistance protein in blast cells from patients with acute leukemia. Blood 2000; 96: 365–68. 46 Hoffmann U, Materna V, Blohmer J-U, et al. Quantitative expression analysis of genes involved in the development of chemoresistance in ovarian carcinoma. Pathol Res Pract 2000; 196: 235. 47 Yang CH, Schneider E, Kuo ML, Volk EL, Rocchi E, Chen YC. BCRP/MXR/ABCP expression in topotecan-resistant human breast carcinoma cells. Biochem Pharmacol 2000; 60: 831–37. 48 Sinha P, Hutter G, Kottgen E, Dietel M, Schadendorf D, Lage H. Search for novel proteins involved in the development of chemoresistance in colorectal cancer and fibrosarcoma cells in vitro using two-dimensional electrophoresis, mass spectrometry and microsequencing. Electrophoresis 1999; 20: 2961–69. 49 Lage H, Jordan A, Scholz R, Dietel M. Thermosensitivity of multidrug-resistant human gastric and pancreatic carcinoma cells. Int J Hyperthermia 2000; 16: 291–303. 50 Futscher BW, Abbaszadegan MR, Domann F, Dalton WS. Analysis of MRP mRNA in mitoxantrone-selected, multidrug-resistant human tumor cells. Biochem Pharmacol 1994; 47: 1601–06. 51 Maliepaard M, van Gastelen MA, de Jong LA, et al. Overexpression of the BCRP/MXR/ABCP gene in a topotecan-selected ovarian tumor cell line. Cancer Res 1999; 59: 4559–63.

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