Leukemia (1999) 13, 578–584 1999 Stockton Press All rights reserved 0887-6924/99 $12.00 http://www.stockton-press.co.uk/leu Role of MRP1 in multidrug resistance in acute myeloid leukemia O Legrand, R Zittoun and J-P Marie EA1529, Universite´ Paris 6, Formation de Recherche Claude Bernard, and Service d’He´matologie, Hoˆpital Hoˆtel-Dieu, AP-HP, Paris, France The best characterized resistance mechanism in adult acute drugs among which are doxorubicin, daunorubicin, idarub- myeloid leukemia (AML) is the one mediated by the MDR1 gene icin, mitoxantrone and etoposide.13,14 At least in transfectants, which has been shown to be associated with poor outcome. However, alternative proteins such as the more recently recog- MRP1 is able to act as an energy-dependent drug pump, nized multidrug-associated protein (MRP1), may also contrib- mainly localized in the plasma membrane, that extrudes drugs ute to the resistance to anthracyclines and etoposide in AML. from the cell against a concentration gradient.14–17 It has been Recently, the role of this protein was discussed and was shown that MRP1 is identical to a GSH-S conjugate carrier unclear in AML. However, recent data concerning the func- described in many cells, because the endogenous leukotriene tionality and the modulation of the activity of MRP1 may eluci- C4 and other GSH-S conjugates are transported by MRP1.18–22 date its role in comparison with other mechanisms of resist- ance. In this paper, we will review these recent data concerning Recently, it was shown that ATP-dependent uptake of the the role of MRP1 in adult AML. unmodified drug vincristine by membrane vesicles derived Keywords: MDR; MRP1; acute myeloid leukemia; functional test; from MRP1 transfected HeLa cells is dependent on the pres- prognostic factor ence of GSH. It is possible that GSH interacts directly with MRP1 and that this interaction is necessary for transport. In addition, GSH depletion by buthionine sulphoximine (BSO) Introduction increases the accumulation of DNR and Rhodamine 123 (Rh123) in several MRP1-positive cell lines.21 Therefore, The use of the first cytostatic agents produced a dramatic MRP1 is a multiple organic anion transporter, it transports cer- improvement in the management of acute myeloid leukemia tain glutathione conjugates and may be partly dependent on (AML), but drug resistance eventually arose during chemo- intracellular glutathione levels for the transport of anthracy- therapy. Among different mechanisms of resistance, the clines. Marbeuf-Gueye et al23 studied the transport kinetics of appearance of the multidrug resistance (MDR) phenotype is a series of anthracyclines in tumor cell lines that overexpress the most frequently observed in many cancer cell lines MRP1 and Pgp. They showed that the transport kinetics of (including leukemia). The best characterized resistance mech- anthracyclines by MRP1 and Pgp are very similar except for anism is the one mediated by the MDR1 gene which has been OH-DOX and OH-DNR which had a lower VMax (maximal shown to be associated with poor outcome.1 However, alter- efflux rate) in the case of MRP1-mediated transport suggesting native proteins such as the multidrug-associated protein a role for the amino group in the interaction with glutathione. (MRP1), may also contribute to the resistance to anthracy- On the other hand, Lorico et al24 compared the sensitivity clines and etoposide in AML.2,3 The role of MRP1 is still being to chemotherapeutic drugs and toxins of wild-type W9.5 discussed and is unclear in AML.4–11 In this paper, we will embryonic stem cells and of single and double MRP1 gene review recent data concerning the role of MRP1 in adult AML. knockout cells derived there from. MRP1 expression was tot- ally abrogated in the double knockout cell line and partially abrogated in the single knockout cell line. RT-PCR analyses Structure and function of MRP1 demonstrated that MDR1 gene was not expressed. The cyto- toxic activities of etoposide, teniposide, vincristine, doxorub- The MRP1 gene is located on chromosome 16p13.1. MRP1, icin, daunorubicin and sodium arsenite were significantly like Pgp belongs to the ATP binding cassette (ABC) super- greater in double knockout cells than in parenteral wild-type family of membrane transport protein. It is a glycosylated ES cells. These findings indicate that baseline MRP1 transmembrane protein of 190 kDa.2 Bakos et al’s12 experi- expression has the capacity to exert a protective role against ment and the alignment of the MRP1 sequence with the the toxicity of multiple chemotherapeutic agents and natural human cystic fibrosis transmembrane conductance (CFTR) toxins. In the same way, deletion of the MRP1 gene was found suggest that human MRP1 contains a tandem repeat of six previously in five of 13 AML patients with inv(16) and was transmembrane helices, each followed by a nucleotide bind- associated with longer overall survival and disease-free sur- ing domain and that the C-terminal membrane-bound region vival.25 This aberration was suggested as being the reason for is glycosylated. The N-terminal region of MRP1 contains an the better prognosis of patients with inv(16) in this small series additional membrane-bound, glycosylated area with four or of patients. However, lack of MDR1 gene expression might five transmembrane helices, which seems to be a character- be an alternative explanation for the good prognosis of these istic feature of MRP-like ATP-binding cassette transporters. patients with inv(16).25 Transfection experiments with different eukaryotic Zaman et al26 have shown that in the MRP-overexpressing expression vectors containing full-length complementary cell line GLC4/ADR the MRP1 gene copy number is increased DNAs of the MRP1 gene have shown that MRP1 can confer, about 25-fold, as compared to the parental cell line. However, just like Pgp, resistance to a broad range of natural product no AML patients analyzed by Burger et al,27,28 including those who had high MRP mRNA expression showed evidence for MRP1 gene amplification. Therefore, they concluded that the increase in MRP1 expression in AML patients was not associa- Correspondence: O Legrand, Hoˆpital Hoˆtel Dieu, 1 place du parvis Notre Dame, Service d’he´matologie, 75 181 Paris Cedex 04, France; ted with amplification of the cognate gene. 29 Fax: 33 1 42 34 82 54 Ma et al demonstrated that MRP1 is highly phosphoryl- Received 29 September 1998; accepted 9 December 1998 ated and that phosphate groups are metabolically active and MRP1 in AML O Legrand et al 579 undergo cycles of phosphorylation and dephosphorylation in bone marrow cells, while we have shown that MRP1 activity the cell. Serine is the single amino acid phosphorylated in was higher in CD34− than in CD34+ leukemia cells.11 MRP1. The effect of H-7 and staurosporine (two protein kinase Several studies on MRP1 expression in AML have been inhibitors) can reduce the phosphorylation of this protein. It reported. The incidence of MRP1 gene overexpression in AML has also been found that, in the presence of these agents, there patients was obtained by RNA or protein techniques. Messen- is a major increase in drug accumulation and concomitant ger RNA was quantified using either RT-PCR or by RNase pro- inhibition in drug efflux of resistant cells. Therefore, certain tection assay. Protein was recently detected by immunocyto- phosphate groups of protein MRP1 play an important role in chemistry, Western blot and flow cytometry with monoclonal modulating drug accumulation in resistant cells. antibodies (MRPm6, MRPr1, QRL-1 and QRL-3).4,6–8,10,11,28,39–43 The DNA containing promoter activity has been sequenced All these antibodies recognize an internal epitope of in its entirety and found to contain multiple putative regulat- MRP1.16,17 Few comparative analyses between these tech- ory sites. Studies using a number of different deletion mutants niques have been published. Filipits et al44 showed that there suggest that the major promoter activity of MRP1 resides in a was a fair correlation between results obtained with QCRL- highly GC-rich region of 914 nucleotides (nucleosides −91 to 1/QCRL-3 and those obtained with MRPr1 by immunocytoch- +103). Promoter activity contained within this sequence, how- emistry for AML patients. In other studies we have demon- ever, is modulated by both positive and negative regulatory strated that three techniques (RT-PCR, in situ hybridation and elements.30 The functional analysis of the nucleotide binding protein detection by flow cytometry) gave similar results in domains of MRP1 identify several mutations that are critical normal hematopoietic cells and in fresh leukemic cells.11,39 for MRP-mediated multidrug resistance.31 Recently, besides MRP1 at least four other homologues of Analyses of the subcellular distribution of MRP1 in cell lines these proteins (these new genes were called MRP2 to MRP5) revealed that approximatively 80% of MRP1 in the SW-1573 have been identified.45 However, MRP3 and MRP5 were only and HeLa transfectants were associated with plasma mem- overexpressed in a few cell lines, and the RNA levels did not brane, while the comparable localization in the drug-selected seem to correlate with resistance to either doxorubicin or cis- H69AR cells contained only approximatively 50% of the pro- platin. MRP4 was not overexpressed in any of the cell lines tein. The remaining MRP1 was codistributed in endocytotic which were analyzed. MRP2 (or cMOAT) was substantially vesicles. The relatively high proportion of MRP1 associated overexpressed in several cell lines, and MRP2 RNA levels cor- with these fractions in H69AR cells may contribute to the lack relate with cisplatin but not doxorubicin resistance in a subset of an observable accumulation defect in these cells when of resistant cell lines. Sequence analysis revealed that the pro- compared with the transfectants.2 In AML cells MRP1 staining tein segment used to generate the MRPm6 antibody was (by immunocytochemistery) was primarily cytoplasmic.32 derived from the most homologous portion located in the C- Several publications have shown that the resistance of cell terminal part of MRP1.