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Drug Metab. Pharmacokin. 18 (3): 194–202 (2003).

Regular Article Expression and Functional Characterization of Human ABC Transporter ABCG2 Variants in Insect Cells

Toshihisa ISHIKAWA1, Shiho KASAMATSU1,YukoHAGIWARA1,HideyukiMITOMO1, Ryo KATO1 and Yasuhiro SUMINO2,3 1Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Kanagawa, Japan 2Strategic Product Planning Department, Takeda Chemical Industries, LTD., Osaka, Japan 3Pharma SNP Consortium, The Japan Pharmaceutical Manufacturer's Association (JPMA), Tokyo, Japan

Summary: Hitherto three variant forms of ABCG2 have been documented on the basis of their amino acid moieties (i.e., Arg, Gly, and Thr) at the position 482. In the present study, we have generated those variants of ABCG2 by site-directed mutagenesis and expressed them in Sf9 insect cells. The apparent molecular weight of the expressed ABCG2 variants was 130,000 under non-reductive conditions, whereas it was reduced to 65,000 by treatment with mercaptoethanol. It is suggested that ABCG2 exists in the plasma membrane of Sf9 cells as a homodimer bound through cysteinyl disulˆde bond(s). Both ATPase activity and drug transport of ABCG2 variants were examined by using plasma membrane fractions prepared from ABCG2-overexpressing Sf9 cells. The ATPase activity of the plasma membrane expressing ABCG2 (Gly-482) was signiˆcantly enhanced by prazosin. In contrast, ABCG2 (Arg-482) transports [3H] in an ATP-dependent manner; however, no transport activity was observed with the other variants (Gly-482 and Thr-482). It is strongly suggested that the amino acid moiety at the position of 482 is critical for the substrate speciˆcity of ABCG2.

Key words: ABC transporter; ABCG2; BCRP; single nucleotide polymorphism; pharmacogenomics

multiple factors involved in its cause and progression.2) Introduction Despite enormous costs and eŠorts spent on the de- There is accumulating evidence that individual velopment of cancer , anticancer drugs variations in response to a drug originate from diŠerent are often eŠective only in a relatively small proportion causes, such as genetic polymorphism and altered ex- of cancer patients. It has long been recognized that the pression levels of drug target molecules (e.g., membrane eŠectiveness of anticancer drugs can vary signiˆcantly receptors, nuclear receptors, and enzymes) as well as among individual patients. Indeed, acquired and intrin- those of drug metabolizing enzymes and drug transport- sicdrugresistanceincanceristhemajorobstacleto ers.1) It is critically important that we understand the long-term, sustained patient response to . relationship between the genetic alterations and the There is accumulating evidence that active export of molecular mechanisms underlying such variations in anticancer drugs from cancer cells is one of the major drug response. Pharmacogenetic and pharmacogenomic mechanisms of drug resistance. Several ATP-binding approaches are expected to signiˆcantly contribute to cassette (ABC) transporters underlie multidrug the realization of ``personalized medicine'' in the near resistance in cancer cells by actively extruding the future. clinically administered chemotherapeutic drugs. Two Cancer is one of the gene-associated diseases, with major ABC transporters, ABCB1 (P-glycoprotein or

Received; April 9, 2003, Accepted; May 12, 2003 To whom correspondence should be addressed: Toshihisa ISHIKAWA,Ph.D.,Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta 4259, Yokohama, Kanagawa 226-8501, Japan. Tel. ＋81-45-924-5800, Fax. ＋81-45-924-5838, E-mail: tishikaw＠bio.titech.ac.jp The present study was supported by research grants entitled ``Studies on the genetic polymorphism and function of pharmacokinetics-related proteins in Japanese population'' (H12-Genome-026) and ``Toxicoproteomics: Expression of ABC transporter genes and drug-drug interactions'' (H14-Toxico-002) from the Japanese Ministry of Health and Welfare as well as by a Grant-in-Aid for Creative Scientiˆc Research (No. 13NP0401) and a research grant (No. 14370754) from the Japan Society for the Promotion of Science. In addition, this study was supported, in part, by the institutional core grant of the 21st Century COE Program, the Ministry of Education, Culture, Sports, Science and Technology.

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MDR1) and ABCC1 (MRP1), have been well studied in 3?, where the primers were designed based on the terms of their structure and function in cancer drug ABCG2 cDNA sequences. The PCR reaction consisted resistance.3–8) In addition, a novel ABC transporter, of 30 cycles of 959C for 30 sec, 589Cfor30sec,and breast cancer resistant protein (BCRP), has recently 729C for 2 min. The resulting PCR product was inserted been discovered in -resistant breast cancer into the pCR2.1 TOPO vector, and its sequences were cells.9) The same transporter has also been found in analyzed by automated DNA sequencing (TOYOBO human placenta10) as well as in drug-resistant cancer cell Gene Analysis, Tokyo, Japan). The open reading frame selected and DNA I inhibi- of the ABCG2 cDNA was identical to the ABCG2 wild tors.11–18) The newly found ABC transporter protein is type (Arg-482) originally named ABCP (GenBank now named ABCG2 and is classiˆed in the G-subfamily accession number: AF103796). The ABCG2 (Arg-482) of human ABC transporter genes according to the new cDNA was removed from the pCR2.1 TOPO vector by nomenclature. The G-subfamily is a so-called ``half- EcoRI digestion. After the treatment with alkaline transporter'' bearing six transmembrane domains and phosphatase, ABCG2 cDNA was ligated to the EcoRI one ATP-binding cassette. site of the pFASTBAC1 Expression vector (Invitrogen) Overexpression of ABCG2 reportedly confers cancer using the Rapid DNA ligation kit (Roche Diagnosis Co., cell resistance to anticancer drugs, such as mitoxan- Indianapolis, IN, USA). trone, , and 7-ethyl-10-hydroxycamptothecin Generation of variant forms by site-directed (SN-38: active metabolite of ). SN-38-selected mutagenesis: The pFASTBAC1 Expression vector PC-6WSN2-5H human lung carcinoma cells were shown carrying the ABCG2 (Arg-482) cDNA was used as the to overexpress ABCG2 with the reduced intracellular ac- template, and variant forms (Gly-482 and Thr-482) were cumulation of SN-38 and its glucuronide metabolite.19) created by the site-directed mutagenesis (Fig. 1B) We have recently demonstrated that plasma membrane using the QuickChange Site-directed Mutagensis Kit vesicles prepared from those cells ATP-dependently (Stratagene) and internal complementary PCR primers transported both SN-38 and SN-38-glucuronide, and as follows: 5?-CTGATTTATTACCCATGGGGATGT- our results strongly suggested that ABCG2 is involved in TACCAAGTATT-3? and 5?-AATACTTGGTAACAT- the active extrusion of SN-38 and its metabolite from CCCCATGGGTAATAAATCAG-3? (for the Gly-482 cancer cells.20) variant form) or 5?-CTGATTTATTACCCATGACGA- To date, at least three variant forms of ABCG2 have TGTTACCAAGTATT-3? and 5?-AATACTTGGTAA- been documented on the basis of amino acid moieties at CATCGTCATGGGTAATAAATCAG-3? (for the position 482, which is located in the third transmem- Thr-482 variant form). The PCR reaction consisted 16 brane domain (Fig. 1). The wild type form of ABCG2 cycles of 959Cfor30sec,559Cfor1minand689Cfor has an arginine (Arg) at that position,10) whereas other 15 min, and Pfu Turbo DNA polymerase was used for variants cloned from cancer cell lines9,13) have glycine the PCR reaction. The mutations were conˆrmed by (Gly) and threonine (Thr) at position 482. It is currently sequencing the inserted fragments. The cDNAs of speculated that the substrate speciˆcities of ABCG2 ABCG2 Gly-482 and Thr-482 variants, thus obtained, may diŠer among those variant forms.18,21–23) To eluci- were separately inserted into the original pFASTBAC1 date the role of amino acid moieties at position 482 in Expression vector (Invitrogen), as described above. the transport function, we have expressed each variant Expression of the human ABCG2 cDNA in Sf9 cells form of ABCG2 in Sf9 insect cells and examined the and preparation of cell membranes: Recombinant activity of those variants. Using the insect cell system, baculoviruses to express the above-mentioned variant we provide direct evidence that the human ABCG2 forms of ABCG2 in insect cells were generated with the protein functions as a homodimer bound via a cysteinyl BAC-TO-BAC Baculovirus Expression Systems (In- disulˆde bond(s). Furthermore, in the present study, we vitrogen) according to the manufacture's instruction. demonstrate that Arg-482 is critically involved in the Insect Spodoptera frugiperda Sf9 cells (1×106 cell WmL) substrate speciˆcity of ABCG2. were infected with the recombinant baculoviruses and cultured in the EX-CELLTM 420 Insect serum-free Methods medium (JRH Bioscience, Levexa, KS, USA) at 269C Cloning of human ABCG2 (Arg-482) cDNA: Hu- with gentle shaking. 48 hours after the infection, cells man ABCG2 cDNA was cloned from mRNA of the were harvested by centrifugation. Cell membranes were MCF7WBCRP clone-8 cell line (Mitomo, H. et al., prepared as described previously.24) After the measure- unpublished work). RT-PCR was carried out by using ment of protein concentration by the BCA Protein the SuperScript First-Strand Synthesis System (Invitro- Assay Kit (PIERCE, Rockford, IL, USA), the mem- gen, Carlsbad, CA, USA) and the following speciˆc brane preparation was stored at －809C until used. primers: sense 5?-CTCTCCAGATGTCTTCCAGT-3? Immunological detection of ABCG2 protein: and antisense 5?-ACAGTGTGATGGCAAGGGAAC- Expression of ABCG2 in Sf9 cell membranes was deter- p194 p.3 [100%]

196 Toshihisa ISHIKAWA, et al.

Fig. 1. Schematic illustrations of the molecular structure of ABCG2 (A) and the pFASTBAC vector for the expression of ABCG2 variants in Sf9 cells (B). A: The molecular structure of ABCG2 was predicted from the cDNA sequence (AF103796) by using the SOSUI program (http://sosui.proteome.bio.tuat.ac.jpWsosuimenu0.html, and the amino acid 482 is indicated by an arrow. B: The partial cDNA sequences of three variants (Arg-482, Gly-482, and Thr-482) are shown in the recombinant expression vector pFASTBAC1 with the ABCG2 cDNA insert (B).

mined by immunoblotting with BXP-21 (SIGNET, Reagent Plus (PerkinElmer Life Sciences, Boston, MA, Dedham, MA, USA), a speciˆc antibody to human USA) and detected by Lumino Imaging Analyzer ABCG2 where membrane proteins were pre-treated with FAS-1000 (TOYOBO, Osaka, Japan). or without mercaptoethanol. Brie‰y, proteins of the ATPase activity measurement: The ATPase activity isolated plasma membrane were separated by elec- of the isolated Sf9 cell membrane was determined by trophoresis on 7.5z sodium dodecyl sulfate (SDS) poly- measuring inorganic phosphate liberation26) according acrylamide slab gels,25) and the proteins were electrob- to the procedure reported by Sarkadi et al.27) with some lotted onto Hy-bond ECL nitrocellulose membranes modiˆcations. To adapt to our high throughput screen- (Amersham, Buckinghamshire, UK). Immunoblotting ing system with 96-well plates, we developed the stan- was performed by using BXP-21 (1:250 dilution) as the dard procedure, as described previously.24) ˆrst antibody and an anti-mouse IgG-horseradish Detection of ATP-dependent transport of peroxidase (HRP)-conjugate (Cell Signaling Technolo- [3H]methotrexate: The frozen stocked membrane was gy, Beverly, MA, USA)(1:3000 dilution) as the sec- thawed quickly at 379C, and vesicles were formed by ondary antibody. HRP-dependent luminescence was passing the membrane suspension through a 27-gauge developed by using Western Lighting Chemiluminescent needle. The standard incubation medium contained p194 p.4 [100%]

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is strongly suggested that ABCG2 exists in the plasma membrane of Sf9 cells as a homodimer bound through cysteinyl disulˆde bond(s). Drug-stimulated ATPase activity in ABCG2-express- ing cell membranes: Using membranes prepared from ABCG2-expressing Sf9 cells, we have measured ATPase activity in the presence of various drug compounds, i.e., prazosin, mitoxantron, and verapamil. As demonstrat- ed in Fig. 3A, prazosin signiˆcantly enhanced the ATPase activity of the plasma membrane prepared from ABCG2 (Gly-482)-expressing Sf9 cells. The stimu- lation of ATPase activity was dependent on the concen- tration of prazosin, exhibiting a saturation curve with a half-maximal concentration of 5 mM. In addition, mitoxantron (50 or 100 mM) enhanced the ATPase activity in the membrane, however its extents were relatively small (Fig. 3A). On the other hand, in the membrane from ABCG2 (Thr-482)-expressing Sf9 cells, the ATPase activity was stimulated by prazosin to a certain extent, but not signiˆcantly by mitoxantron. Fig. 2. EŠect of mercaptoethanol on dimerization of ABCG2. The plasma membrane from ABCG2 (Arg-482)-transfected Sf9 cells Interestingly, no stimulation was observed with prazo- was incubated with or without 100 mM mercaptoethanol at room tem- sin or mitoxantron in the membrane from ABCG2 (Arg- perature for 20 min. Thereafter, membrane proteins (2 mgforeach 482)-expressing Sf9 cells. Verapamil, which is a sub- lane) were separated by SDS-PAGE and ABCG2 was immunological- strate for ABCB1 (P-glycoprotein, MDR1) but not for ly detected by Western blotting as described in Methods. ABCG2,hadnoeŠectontheATPaseactivityofthe membranes prepared from those three variant forms- expressing Sf9 cells. plasma membrane vesicles (40 or 80 mg of protein), EŠect of vanadate on prazosin-stimulated ATPase 10 mM [3?,5?,7?-3H]methotrexate (Amersham, Buckin- activity: As shown in Fig. 3B, prazosin-stimulated ghamshire, UK), 0.25 M sucrose, 10 mM TrisWHCl, ATPase activity in the ABCG2 (Gly-482)-expressing

pH 7.4, 10 mM MgCl2, 1 mM ATP, 10 mM creatine membrane was sensitive to vanadate, where ATPase phosphate, and 100 mgWmL creatine kinase in a ˆnal activity was almost completely inhibited by 50 mM sodi- volume of 110 mL. The reaction was started by adding um vanadate to background levels. Likewise, in the [3H]methotrexate to the incubation medium. The reac- ABCG2 (Thr-482)-expressing membrane, prazosin- tion was carried out at 379C, and the amount of stimulated ATPase activity was inhibited by vanadate in [3H]methotrexate incorporated into the vesicles was a similar dose-dependent manner. In the absence of measured by a rapid ˆltration technique as previously prazosin, the basal ATPase activity was little aŠected by described.28) vanadate in the concentration range of up to 200 mM. This suggests that prazosin-stimulated ATPase activity Results can be distinguished from the basal ATPase activity by Expression of ABCG2 variant forms in Sf9 cells: such vanadate eŠects. Three variant forms (i.e., Arg-482, Gly-482, and ATP-dependent transport of methotrexate by Thr-482) of human ABCG2 were generated by site- ABCG2 (Arg-482): The transport function of directed mutagenesis and expressed at high levels in Sf9 Arg-482, Gly-482, and Thr-482 variants was examined cells by infection with the baculoviruses derived from by using plasma membrane vesicles prepared from Sf9 the recombinant pFASTBAC1 expression vectors cells expressing those variants. Figure 4A depicts the (Fig. 1B). The expression of ABCG2 proteins in the time courses of methotrexate transport into plasma plasma membrane of Sf9 cells was detected by Western membrane vesicles in the presence or absence of ATP. blotting. As demonstrated in Fig. 2, the molecular size The ATP concentration was maintained at constant of ABCG2 expressed in Sf9 cells was 65,000 under levels for a period su‹cient for measurement (at least reductive conditions (with mercaptoethanol), whereas 20 min) with the creatine phosphate and creatine kinase its apparent molecular weight was 130,000 under non- reaction system. It is important to note that ATP- reductive conditions (without mercaptoethanol) dependent methotrexate transport was observed only in (Fig. 2). This phenomenon was observed equally for the plasma membrane vesicles prepared from Sf9 cells Arg-482, Gly-482 and Thr-482 variants, and therefore it expressing the Arg-482 variant. No signiˆcant transport p194 p.5 [100%]

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Fig. 3. EŠect of prazosin, mitoxantron, and verapamil (A) as well as vanadate (B) on the ATPase activity of cell membranes prepared from Sf9 cells. The ATPase activity was measured as described in the section of Methods. A: Drugs, i.e., prazosin (), mitoxantron (), and verapamil (), were added to the reaction mixture at diŠerent concentrations as indicated. B: To test the inhibitory eŠect of vanadate, the ATPase activity was measured in the absence () and presence ()of50mM prozosin. Results are expressed as mean values±S.D. in triplicate measurements.

activity was detected from the Gly-482 or Thr-482 Discussion variant-expressing Sf9 cells. Figure 4B demonstrates a relationship between the methotrexate concentration Expression of human ABCG2 as a homodimer in and the rate of ATP-dependent transport by the insect cells: Since ABCG2 is an ABC half-transporter, Arg-482 variant. Based on the Lineweaver-Burk plot of it has been suspected that ABCG2 functions as a this result, the apparent Km value for methotrexate was homodimer or heterodimer.15) Most recently, using estimatedtobe6mM. cross-linking reagents and speciˆc antibodies, Litman et al. have provided evidence for the formation of an p194 p.6 [100%]

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Fig. 4. ATP-dependent transport of methotrexate (MTX) in plasma membrane vesicles prepared from ABCG2-transfected Sf9 cells (A) and the eŠect of MTX concentration on the transport (B). A: Plasma membrane vesicles were prepared from mock- and ABCG2 (Arg-482, Gly-482, or Thr-482)-expressing Sf9 insect cells as described in Experimental. Plasma membrane vesicles (80 mg of protein) were incubated with 10 mM [3H]methotrexate (MTX) in the absence () or presence

() of 1 mM ATP in the medium containing 0.25 M sucrose, 10 mM TrisWHCl, pH 7.4, 10 mM MgCl2, 10 mM creatine phosphate, and 100 mgW mL creatine kinase. The incubation was carried out at 379C and then stopped at diŠerent time points (0, 5, 10 15 and 20 min) as indicated in the ˆgure. The amount of [3H]methotrexate incorporated into the membrane vesicles was measured by a rapid ˆltration technique. Results are expressed as mean values±SD in triplicate measurements. B: The eŠect of the MTX concentration on the initial rate of ATP-dependent MTX transport into membrane vesicles. The ATP-dependent MTX transport was measured as described above, and the initial rate was measured at 0¿20 min in the incubation.

ABCG2 homodimer.29) In addition, Kage et al.have dimerization through cysteinyl disulˆde bonds occurred recently established PA317 transfectants expressing in all three variant forms of human ABCG2 tested in Myc and HA epitope-tagged ABCG2 proteins, and they this study. Similar results were obtained with human demonstrated that those hybrid proteins formed S-S ABCG2 expressed in human embryonic kidney (HEK) homodimers.30) In the present study, by expressing 293 cells (Mitomo et al., manuscript submitted). There- native ABCG2 (without tag) in insect cells, we could fore, it is strongly suggested that the human ABCG2 provide direct evidence that human ABCG2 exists in the protein expressed in Sf9 insect cells operates as a plasma membrane as a homodimer bound through homodimer as does the protein expressed in mammalian cysteinyl disulˆde bond(s) (Fig. 2). Treatment with cells. This knowledge is important, since ABCG2 ex- mercaptoethanol reduced the apparent molecular pressed in Sf9 cells may be widely used for the function- weight of ABCG2 from 130,000 to 65,000 (Fig. 2). The al screening of ABCG2 with drug candidates. Based on p194 p.7 [100%]

200 Toshihisa ISHIKAWA, et al.

the cDNA sequence, a total of twelve cysteine residues underlying the drug resistance should be rather com- exist in the ABCG2 peptide. From a biochemical point plex. The actual involevement of ABCG2 (Arg-482) in of view, it is of great interest to study which cysteine the methotrexate resistance remained to be elucidated. residues participate in the disulˆde bond formation and In the present study using the plasma membrane vesicle how inter-peptide disulˆde bonds are formed in the cell. system, we could clearly demonstrate that ABCG2 Drug-stimulated ATPase activity of ABCG2 variants: (Arg-482) transports methotrexate; but, the other Several acquired mutations have hitherto been variants Gly-482 and Thr-482 do not (Fig. 4). Our ˆnd- documented for ABCG2 cloned from drug resistant cell ing suggests that the presence of Arg-482 in the third lines.21,31) Drug resistance phenotypes vary among diŠer- transmembrane domain is a critical amino acid residue ent cell lines expressing variant types of ABCG2. In involved in the substrate speciˆcity of ABCG2. Since fact, transfectants with the wild type (Arg-482) were not the arginine residue is positively charged under the phys- resistant to topotecan,18) while overexpression of the iological condition, it is likely that its positive charge is a Gly-482 and Thr-482 variants conferred resistance to prerequisite for interactions between the active site of mitoxantrone, doxorubicin, , and various ABCG2 and anionic substrates, such as methotrexate. analogs including topotecan.9,14–16) Earlier works32,33) have predicted the existence of ener- Furthermore, Gly-482 and Thr-482 variants mediated gy-dependent transport systems for the methotrexate the eŒux of rhodamine 123 and doxorubicin from cells; eŒux from cells. Molecular cloning and functional however, Arg-482 did not.21,22) These ˆndings indicate expression of ABC transporters led to the identiˆcation that Arg at position 482 plays a critical role in the of human ABCC1, ABCC2, ABCC3, and ABCC4 as substrate speciˆcity of ABCG2. The identiˆcation of methotrexate transporters,34–36) and all of these trans- mutations at 482 in ABCG2 may explain some dis- porters belong to the C-subfamily of human ABC trans- crepancies observed in the cross-resistance proˆles of porters.7,8) In this context, this is the ˆrst report that human cancer cell lines. To examine the function of ABCG2, a member of the ``G'' sub-family, transports ABCG2 as a drug transporter, we have previously estab- methotrexate. However, because of its high Km value of lished ABCG2 (Arg-482)-overexpressing Sf9 insect cells ABCG2 to methtrexate (6 mM; Fig. 4B), the contribu- and tried to measure drug-induced ATPase activity.24) tion of this transporter to methotrexate resistance might However,becauseofhighlevelsofthebasalATPase be small at physiological concentrations. A relevant activity present in the plasma membrane, we were not study on the relationship between ABCG2 variant forms able to accurately measure the drug-induced ATPase expressed in HEK293 cells and their drug resistance activity of ABCG2.24) proˆles will be reported elsewhere.37) In the present study, prazosin was found to sig- Concluding Remarks: Like human ABCG2, one niˆcantly enhance the ATPase activity in plasma mem- mutation `hot spot' was identiˆed at the same amino branes prepared from Gly-482 and Thr-482 variants- acid position 482 in mouse Abcg2, where the Arg-482 is expressing Sf9 cells. However, such stimulation was not the wild type.38) In the case of mouse Abcg2, variants of observed in the plasma membrane expressing the Arg, Ser, and Met signiˆcantly aŠect the drug resistance Arg-482 variant (Fig. 3A). The prazosin-stimulated proˆle in cancer cell lines.38) Recently, Jonker et al. ATPase activity was dose-dependently inhibited by demonstrated that mouse Abcg2 protects against a

vanadate (IC50＝20 mM; Fig. 3B). Such inhibition by major chlorophyll-derived dietary phototoxin and pro- vanadate is characteristic for ABC transporters, includ- toporphyria.39) In their study, the wild type (Arg-482) of ing ABCB1 (P-glycoproteinWMDR1)24) and ABCC1 mouse Abcg2 has been suggested to transport chlo- (MRP1WGS-X pump).28) Thus, it is suggested that rophyll and porphyrin metaboites. Since those metabo- Gly-482 and Thr-482 variants of human ABCG2 trans- lites are organic anions like methotrexate, it is hypothe- port prazosin, but the Arg-482 variant does not or its sized from our ˆndings that the Arg-482 moiety may be transport activity is very low. In several cases, however, important for the transport of such anionic metabolites. the substrate stimulation of the ABCG2-ATPase could With this respect, further studies should be needed to not be directly correlated with the actual transport examine our hypothesis. process, as suggested byOzvegy Ä et al.22) It would be important to examine the actual transport of drugs by Acknowledgements: The authors thank the Pharma ABCG2 variant forms. SNP Consortium (PSC) for generous support through- ATP-dependent methotrexate transport by the out this study. ABCG2 (Arg-482): Recently, Volk et al.suggested References that overexpression of the wild type (Arg-482) of ABCG2 mediates methotrexate resistance.23) However, 1) Kalow,W.,Meyer,U.A.andTyndale,R.F.(eds.): the mitoxantrone-selected MCFWMX cell line was used Pharmacogenomics, Marcel Dekker, New York (2001). in their study, and therefore molecular mechanisms 2) Hesketh, R.: The Oncogene Handbook,Academic p194 p.8 [100%]

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Press, London (1994). Natl. Cancer Inst., 91: 429–433 (1999). 3) Ling, V.: Multidrug resistance: molecular mechanisms 16) Brangi, M., Litman, T., Ciotti, M., Nishiyama, K., and clinical relevance. Cancer Chemother. Pharmacol., Kohlhagen, G., Takimoto, C., Robey, R., Pommier, Y., 40: S3–S8 (1997). Fojo, T. and Bates, S. E.: Camptothecin resistance: role 4) Ambudkar, S. V., Dey, S., Hrycyna, C. A., of the ATP-binding cassette (ABC), mitoxantrone- Ramachandra, M., Pastan, I. and Gottesman, M. M.: resistance half-transporter (MXR), and potential for Biochemical, cellular, and pharmacological aspects of glucuronidation in MXR-expressing cells. Cancer Res., the multidrug transporter. Annu. Rev. Pharmacol. 59: 5938–5946 (1999). Toxicol., 39: 361–398 (1999). 17) Litman, T., Brangi, M., Hudson, E., Fetsch, P., Abati, 5) Klein, I., Sarkadi, B. and V áaradi, A.: An inventory of A., Ross, D. D., Miyake, K., Resau, J. H. and Bates, S. the human ABC proteins. Biochim. Biophys. 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