Overexpression of the breast cancer resistance protein (BCRP) efflux pump in human cancer cell lines results in resistance to a variety of cytostatic agents. The aim of this study was to analyze BCRP protein expression and activity in acute myeloid leukemia (AML) samples and to determine whether it is up-regulated due to clonal selection at relapse/refractory disease. BCRP protein expression was measured flow cytometrically with the monoclonal antibodies BXP-34 and BXP-21 in 20 paired samples of de novo and relapsed/refractory AML. BXP-34/immunoglobulin G1 ratios were observed of 1.6 ± 0.5 (mean ± SD, range 0.8-2.7) and BXP-21/immunoglobulin G2a ratios of 4.9 ± 3.0 (range 1.1-14.5) in the patient samples versus 9.8 ± 6.8 and 6.5 ± 2.4, respectively, in the MCF-7 cell line. BCRP activity was determined flow cytometrically by measuring mitoxantrone accumulation in absence and presence of the inhibitor fumitremorgin C. Mitoxantrone accumulation, expressed as mean fluorescence intensity (MFI), varied between 44 and 761 MFI (227 ± 146 MFI) and correlated inversely with BCRP expression (r = −0.58, P < .001). Addition of fumitremorgin C showed a small increase in mitoxantrone accumulation (11 ± 29 MFI, n = 40) apart from the effect of PSC833 and MK-571. No consistent up-regulation of BCRP expression or activity was observed at relapse/refractory disease; some cases showed an increase and other cases a decrease at relapse. Relatively high BCRP expression correlated with immature immunophenotype, as determined by expression of the surface marker CD34 (r = 0.54, P = .001). In conclusion, this study shows that BCRP protein is expressed at low but variable levels in AML, especially in immature CD34+cells. BCRP was not consistently up-regulated in relapsed/refractory AML.

Introduction

The multidrug resistance proteins P-glycoprotein (P-gp)1 and multidrug resistance protein 1 (MRP1),2,3 which are members of the ATP-binding cassette (ABC) family of transport proteins, have been extensively studied. They are both described to be poor prognostic factors in the treatment of acute myeloid leukemia (AML).4-7 An additional member of the ABC transport family is the breast cancer resistance protein (BCRP), also known as MXR, ABCP, or ABCG2, a 655–amino acid protein encoded by the BCRP gene located on chromosome 4q22.8,9 Overexpression of BCRP has been observed in human cancer cell lines selected for resistance with mitoxantrone,10,11 doxorubicin plus verapamil,12 and topotecan.13 Transfection and overexpression of BCRP in the human breast cancer cell line MCF-7 resulted in resistance to the cytostatic agents mitoxantrone, daunorubicin, and topotecan.8 Substrate specificities of BCRP, P-gp, and MRP1 are distinct but overlapping. Daunorubicin and mitoxantrone are transported by BCRP, P-gp, and MRP1,6,14whereas verapamil is especially transported by P-gp, calcein is effluxed only by MRP1, and lysotracker green was reported as a substrate for BCRP.14 Reversal of BCRP-mediated, but not of P-gp– or MRP-mediated multidrug resistance has been described with fumitremorgin C (FTC), which was effective in reversing resistance to mitoxantrone, doxorubicin, and topotecan in multidrug-selected cell lines.15 

AML patients are treated with intensive chemotherapy regimens, including daunorubicin, idarubicin, and mitoxantrone. Despite these intensive regimens, a considerable number of AML patients show resistance to these drugs and do not obtain complete remission or demonstrate a relapse.16-19 Recently, BCRP messenger RNA (mRNA) expression has been found to be expressed at variable levels in blast cells from de novo AML patients.20 So far no data have been published concerning the functional activity of BCRP in AML. In a previous study we have reported that clonal selection of P-gp– or MRP-expressing cells does not play a role at relapse in AML; the observed changes in P-gp and MRP activity in relapse versus primary AML samples were correlated with changes in maturation stage as determined by immune phenotyping.21 In the present study we were interested in whether clonal selection of BCRP-expressing cells might occur in AML at relapse. Therefore, we determined the expression of BCRP protein with flow cytometry using the recently described BXP-34 and BXP-21 monoclonal antibodies22,23 in 20 paired samples of de novo and relapsed AML. The functional activity of BCRP was determined by measuring the capacity to extrude mitoxantrone in the absence or presence of the BCRP inhibitor FTC in cell lines and samples from patients with AML by means of flow cytometry. Because it has been described that BCRP expression in normal hematopoietic cells is restricted to the most primitive cells,23-25 we were interested in a possible correlation between BCRP expression and primitive immunophenotype in AML. Therefore, we determined the expression of the immature CD34+/CD38 and CD34+/CD33 subpopulations and the more mature CD34+/CD33+ and CD34/CD33+ subpopulations of AML cells and correlated the expression of these subpopulations with BCRP protein expression.

Patients, materials, and methods

Cell lines

The drug-sensitive human breast cancer cell line MCF-7, its mitoxantrone-resistant counterpart MCF-7 MR,11 which overexpresses BCRP but not P-gp or MRP1, and its doxorubicin-selected subline MCF-7 Dox40,11 which overexpresses P-gp, were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS). The MCF-7 MR and the MCF-7 Dox40 cell lines were cultured in the presence of 80 nM mitoxantrone and 400 nM doxorubicin, respectively. Cells were cultured in drug-free medium at least 7 days prior to the analyses.

Patients

After informed consent, bone marrow aspirates or peripheral blood were collected from AML patients at the time of diagnosis and relapse or refractory disease, between July 1992 and November 2000 at the University Hospital Groningen. Patients were classified at presentation of the disease according to the French-American-British (FAB) classification. Leukemic blasts were enriched by Ficoll-Isopaque (Nycomed, Oslo, Norway) density gradient centrifugation; cryopreserved in RPMI 1640 medium (BioWhittaker, Brussels, Belgium) supplemented with 10% FCS (Hyclone, Logan, UT) and 10% dimethyl sulfoxide (Merck, Amsterdam, The Netherlands); and stored at −196°C. Paired cases, which were available at the time of diagnosis as well as at relapse, were selected. Upon analysis, AML blasts were thawed, treated with deoxyribonuclease (Boehringer Mannheim, Almere, The Netherlands), washed with RPMI 1640 medium, and incubated for 30 minutes in RPMI 1640 medium supplemented with 10% FCS at 37°C, 5% CO2. If more than 10% T cells were present, as determined by the percentage of CD3+ cells, T lymphocytes were depleted by 2-aminoethylisothiouronium bromide–treated sheep red blood cell rosetting. T-cell sheep red blood cell rosettes were removed by Ficoll-Isopaque density gradient centrifugation. May-Grünwald/Giemsa staining was performed to determine the percentage of blast cells in the AML cells, and in all cases the percentage of blast cells was more than 90%.

Patients were treated with chemotherapy regimens according to the protocol of the Dutch-Belgian Hemato-Oncology Cooperative Group for AML (Hovon 4/4A or Hovon 29).26,27 

Normal bone marrow samples

Normal bone marrow samples were obtained after informed consent from 3 patients who underwent cardiac surgery. Samples were cryopreserved, thawed, and T cells were depleted as described above. Viability of the cells was determined by trypan blue exclusion.

Flow cytometric detection of BCRP protein expression

The BCRP protein expression of the tumor cell lines and AML blasts was measured with the BXP-34 and BXP-21 monoclonal antibodies, which recognize internal epitopes of the BCRP protein. Cells (0.5 × 106) were incubated for 10 minutes at room temperature with fluorescence-activated cell sorter (FACS) lysing solution (Becton Dickinson Immunocytometry Systems, Erembodegem, Belgium) to permeabilize the cell membranes. Then the cells were incubated for 15 minutes at room temperature with 1% goat serum in phosphate-buffered saline (140 mM NaCl, 9.0 mM Na2HPO4.2H2O, 1.3 mM NaH2PO4.2H2O, pH 7.4) containing 2% bovine serum albumin. Thereafter, the cells were incubated for 60 minutes at room temperature with 10 μL BXP-34 or BXP-21 mouse antibodies obtained from hybridoma supernatants or with 10 μL (0.5 μg) immunoglobulin G1 (IgG1) or IgG2a isotype control. Cells were washed with phosphate-buffered saline/bovine serum albumin and incubated for 20 minutes with phycoerythrin-conjugated rabbit antimouse F(ab′)2 fragments. Phycoerythrin fluorescence was measured on a FACSCalibur flow cytometer. BCRP protein expressions were expressed as adjusted for IgG1 or IgG2a control, ie, the ratio of BXP-34/IgG1 or BXP-21/IgG2a antibody fluorescence.

Flow cytometric detection of mitoxantrone accumulation

The ability of tumor cell lines and AML blasts to extrude mitoxantrone in the absence or presence of the BCRP inhibitor FTC,15 the P-gp inhibitor PSC833 (provided by Novartis Pharma, Basel, Switzerland), the leukotriene D4 receptor antagonist and MRP inhibitor MK-571,28 or combinations of these inhibitors was measured in a flow cytometric assay.29Cells (0.5 × 106) were preincubated with the inhibitors for 20 minutes at 37°C, 5% CO2, in the following combinations: RPMI 1640 medium alone (0.5 mL), RPMI 1640 medium plus FTC (10 μM),29 the maximum nontoxic doses of PSC833 (2 μg/mL) and MK-571 (20 μM),30 FTC (10 μM) plus PSC833 (2 μg/mL), FTC (10 μM) plus MK-571 (20 μM), PSC833 (2 μg/mL) plus MK-571 (20 μM) (if sufficient cells were available), or FTC (10 μM) plus PSC833 (2 μg/mL) plus MK-571 (20 μM). Thereafter, mitoxantrone (10 μM) was added and the cells were incubated for 60 minutes at 37°C, 5% CO2. Cells were washed with ice-cold RPMI 1640 medium. Fluorescence of mitoxantrone was analyzed with a FACSCalibur flow cytometer (Becton Dickson Medical Systems, Sharon, MA) equipped with an argon laser. The blast population was gated by forward and side scatter characteristics. The mitoxantrone fluorescence of 5000 gated events was logarithmically measured at a laser excitation wavelength of 635 nm through a 670 nm bandpass filter. Mitoxantrone accumulation was expressed as mean fluorescence intensity (MFI). The effects of the inhibitors were expressed as a shift of MFI of the mitoxantrone accumulation.

Flow cytometric detection of P-gp and MRP activity

Functional activity of the P-gp and MRP transporter proteins was demonstrated as described previously.29 In short, to analyze P-gp activity we used rhodamine 123 (Rh123) (Sigma Chemical, Bornem, Belgium) in combination with the P-gp inhibitor PSC833. To determine MRP (MRP1 and homlogs MRP2 and MRP3) activity, the compound carboxyfluorescein (CF) was used in combination with the MRP inhibitor MK-571. Rh123 and CF fluorescence of 5000 events was measured with a FACSCalibur flow cytometer. The efflux-blocking factors of the inhibitors were expressed as MFI in inhibitor-blocked cells divided by the MFI in unblocked cells.

Immunophenotype

To determine the maturation stage of the AML blasts, phenotyping was performed using monoclonal antibodies against CD34 in combination with CD38 or CD33 or IgG isotype–matched controls21(Becton Dickinson, Mountain View, CA). A total of 0.5 × 106 cells were incubated with 5 μL fluorescein isothiocyanate– or phycoerythrin-labeled mouse monoclonal antibodies for 20 minutes at 4°C, washed with RPMI 1640 medium, and analyzed with a FACSCalibur flow cytometer. The percentages of the following subclasses of cells were determined: CD34+, CD33+, the immature subpopulations of CD34+/CD38 and CD34+/CD33 blasts, and the more mature subclasses of CD34+/CD33+ and CD34/CD33+ blasts.

Statistical analysis

The parametric Student t test and the Mann-WhitneyU and Wilcoxon nonparametric tests were used to calculate significant differences, and correlations were calculated using the Spearman bivariate nonparametric correlation test. Data were expressed as mean ± SD. P values below .05 were considered significant.

Results

Cell lines

BCRP protein expression in cell lines.

First, to test whether the expression of BCRP protein could be measured by flow cytometry, we studied BCRP protein expression in the drug-sensitive and BCRP low-expressing cell line MCF-7 in the mitoxantrone-resistant and BCRP-overexpressing cell line MCF-7 MR and in the P-gp–overexpressing cell line MCF-7 Dox40. BCRP protein expression was measured using the monoclonal antibodies BXP-34 and BXP-21 in conjunction with IgG isotype controls. A BXP-34/IgG1 ratio of 9.8 ± 6.8 (mean ± SD, n = 3) (Table1) was observed in the MCF-7 cell line versus a high expression of 50.6 ± 8.1 (n = 3,P < .05) in the MCF-7 MR cell line; the BXP-21/IgG2a ratio was 6.5 ± 2.4 (n = 4) in the MCF-7 cell line versus 11.3 ± 0.6 (n = 4, P < .05) in the resistant MCF-7 MR cell line. The P-gp–overexpressing MCF-7 Dox40 cell line showed a low level of BCRP expression: a BXP-34/IgG1 ratio of 7.6 ± 0.6 (n = 3) and a BXP-21/IgG2a ratio of 2.3 ± 0.3 (n = 3).

Table 1.

BCRP protein expression in MCF-7 cell lines

 BXP-34/IgG1 BXP21/IgG2a  
MCF-7 S 9.8 ± 6.8 6.5 ± 2.4  
MCF-7 MR 50.6* ± 8.1 11.3* ± 0.6  
MCF-7 Dox40 7.6 ± 0.6 2.3 ± 0.3 
 BXP-34/IgG1 BXP21/IgG2a  
MCF-7 S 9.8 ± 6.8 6.5 ± 2.4  
MCF-7 MR 50.6* ± 8.1 11.3* ± 0.6  
MCF-7 Dox40 7.6 ± 0.6 2.3 ± 0.3 

The results are expressed as MFI ratios and represent mean ± SD of at least 3 experiments.

*

P < .05 compared with MCF-7.

Mitoxantrone accumulation in cell lines.

Then, to determine whether the BCRP protein expression was reflected by the capacity to extrude mitoxantrone, the accumulation of mitoxantrone was measured in the cell lines. The accumulation, expressed as MFI, after exposure to mitoxantrone (10 μM) was 211 ± 15 (n = 4) in the MCF-7 cell line versus 93 ± 36 (n = 3, P < .05) in the MCF-7 MR and 109 ± 41 in the MCF-7 Dox40 (n = 3,P < .05) cell lines. This reflects a high capacity of the MCF-7 MR and MCF-7 Dox40 cell lines to extrude mitoxantrone (Table2 and Figure1). To check whether the efflux of mitoxantrone in the MCF-7 MR cell line was BCRP-mediated, the BCRP inhibitor FTC (10 μM) was added. In the MCF-7 cell line, the presence of FTC caused a change in mitoxantrone accumulation from 211 ± 15 MFI to 298 ± 14 MFI, representing a shift of 86 ± 14 MFI (n = 4). In the MCF-7 MR cell line, the addition of FTC caused a larger change in mitoxantrone accumulation from 93 ± 36 MFI to 341 ± 89 MFI (a shift of 254 ± 54, P < .05), which indicated that the high capacity of the MCF-7 MR cell line to transport mitoxantrone was mediated by BCRP (Table 2 and Figure 1). In the P-gp–overexpressing cell line, MCF-7 Dox40 FTC had a limited effect; a shift of 22 ± 22 MFI was observed. Next, the effect of the P-gp inhibitor PSC833 (2 μg/mL) was determined on the accumulation of mitoxantrone. The addition of PSC833 caused low mitoxantrone accumulation shifts of 52 ± 6 MFI (n = 3) in the MCF-7 cell line and 23 ± 9 MFI (n = 3) in the MCF-7 MR cell line, in contrast to a high shift of 242 ± 93 MFI (n = 3, P < .05) in the P-gp–overexpressing MCF-7 Dox40 cell line. This corresponded with the P-gp activities in these cell lines, as determined by measuring the Rh123 efflux-blocking capacity of PSC833. Low Rh123 efflux-blocking factors by PSC833 of 1.2 ± 0.3 and 1.1 ± 0.2 (n = 3) were observed in the MCF-7 and MCF-7 MR cell lines, respectively, versus a high factor of 20.3 ± 2.0 in MCF-7 Dox40 cell line (n = 3, P < .05). MK-571, an inhibitor of MRP1, showed a shift of 89 ± 14 MFI (n = 4) in the MCF-7, 54 ± 16 MFI (n = 3) in the MCF-7 MR, and 36 ± 39 MFI (n = 3) in the MCF-7 Dox40 cell lines. MRP activity, as determined by the CF efflux-blocking factor of MK-571, was 3.3 ± 0.3 in the MCF-7, 4.6 ± 0.9 in the MCF-7 MR, and 7.6 ± 0.9 in the MCF-7 Dox40 cell lines (n = 3) (Table 2).

Table 2.

Mitoxantrone accumulation studies in MCF-7 and MCF-7 MR cell lines

 Mitoxantrone
accumulation, MFI 
FTC shift, MFI PSC833 shift, MFI P-gp activity MK-571 shift, MFI MRP activity 
MCF-7 211 ± 15 86 ± 14 52 ± 6 1.2 ± 0.3 89 ± 14 3.3 ± 0.3 
MCF-7 MR 93* ± 36 254* ± 54 23 ± 9 1.1 ± 0.2 54 ± 16 4.6 ± 0.9 
MCF-7 Dox40 109* ± 41 22* ± 22 242 ± 93 20.3 ± 2.0 36 ± 39 7.6 ± 0.9 
 Mitoxantrone
accumulation, MFI 
FTC shift, MFI PSC833 shift, MFI P-gp activity MK-571 shift, MFI MRP activity 
MCF-7 211 ± 15 86 ± 14 52 ± 6 1.2 ± 0.3 89 ± 14 3.3 ± 0.3 
MCF-7 MR 93* ± 36 254* ± 54 23 ± 9 1.1 ± 0.2 54 ± 16 4.6 ± 0.9 
MCF-7 Dox40 109* ± 41 22* ± 22 242 ± 93 20.3 ± 2.0 36 ± 39 7.6 ± 0.9 

The results represent mean ± SD of at least 3 experiments. The concentrations used are as follows: mitoxantrone, 10 μM; FTC, 10 μM; PSC833, 2 μg/mL; and MK-571, 20 μM. P-gp activity is expressed as PSC833 efflux-blocking factor of Rh123. MRP activity is expressed as MK-571 efflux-blocking factor of CF.

*

P < .05 compared with MCF-7.

P < .05 compared with MCF-7 and MCF-7 MR.

Fig. 1.

Mitoxantrone accumulation.

Accumulation was measured flow cytometrically in MCF-7 (A) and MCF-7 MR (B) cell lines after 60 minutes of incubation without (blank) or with mitoxantrone (10 μM) in the presence (black) or absence (gray) of 10 μM FTC.

Fig. 1.

Mitoxantrone accumulation.

Accumulation was measured flow cytometrically in MCF-7 (A) and MCF-7 MR (B) cell lines after 60 minutes of incubation without (blank) or with mitoxantrone (10 μM) in the presence (black) or absence (gray) of 10 μM FTC.

Patients

Patient characteristics.

Paired bone marrow or peripheral blood samples were collected from 20 AML patients. The patients were treated with intensive chemotherapy regimens according to the protocol of the Hovon 4/4A or Hovon 29,26,27 which included the chemotherapeutic agents cytosine arabinoside (Ara-C, 200 mg/m2, intravenously, days 1-7), daunorubicin (45 mg/m2, intravenously, days 1-3), or idarubicin (12 mg/m2, intravenously, days 5-7) in the induction cycle of chemotherapy and Ara-C (2 mg/m2, intravenously, days 1-6) plus amsacrine (120 mg/m2, intravenously, days 4-6) in the second induction cycle. Patients with FAB classification M3 received all-trans-retinoic acid. After the 2 induction cycles, patients were to receive a third cycle consisting of mitoxantrone (10 mg/m2, intravenously, days 1-5) plus etoposide (100 mg/m2, intravenously, days 1-5) or receive an autograft transplantation after conditioning treatment with busulfan (4 mg/kg, orally, days 7-4 prior to transplantation) and cyclophosphamide (60 mg/kg, intravenously, days 3-2 prior to transplantation). After treatment 15 AML patients reached complete remission, 4 patients reached partial remission defined as less than 25% of AML blasts in the bone marrow smear, and 1 patient was refractory to the treatment. Patient characteristics are described in Table 3.

Table 3.

Characteristics of AML patients

Patient no. FAB classification Treatment Remission 
M2 Ara-C, ida, amsa, asct CR  
M2 Ara-C, ida, amsa, etop, mito PR  
M5 Ara-C, ida, amsa, etop, mito CR 
M0 Ara-C, ida, amsa, asct CR  
M5 Ara-C, ida, amsa, etop, mito CR  
M1 Ara-C, ida, amsa, asct NR 
M5 Ara-C, dauno, etop, mito CR  
M3 Ara-C, ida, amsa, etop, mito CR  
M2 Ara-C, ida, amsa, asct PR 
10 M4 Ara-C, ida, amsa, etop, mito CR 
11 M1 Ara-C, ida, etop, mito CR  
12 M2 Ara-C, ida, amsa, asct CR  
13 M2 Ara-C, dauno, amsa, etop, mito CR  
14 M2 Ara-C, ida, amsa, asct CR 
15 M4 Ara-C, ida, amsa, etop, mito CR 
16 M2 Ara-C, dauno, amsa, etop, mito PR 
17 M2 Ara-C, etop, mito CR  
18 M4 Ara-C, dauno, asct CR  
19 M2 Ara-C, dauno, amsa, asct CR 
20 M0 Ara-C, dauno, amsa, asct PR 
Patient no. FAB classification Treatment Remission 
M2 Ara-C, ida, amsa, asct CR  
M2 Ara-C, ida, amsa, etop, mito PR  
M5 Ara-C, ida, amsa, etop, mito CR 
M0 Ara-C, ida, amsa, asct CR  
M5 Ara-C, ida, amsa, etop, mito CR  
M1 Ara-C, ida, amsa, asct NR 
M5 Ara-C, dauno, etop, mito CR  
M3 Ara-C, ida, amsa, etop, mito CR  
M2 Ara-C, ida, amsa, asct PR 
10 M4 Ara-C, ida, amsa, etop, mito CR 
11 M1 Ara-C, ida, etop, mito CR  
12 M2 Ara-C, ida, amsa, asct CR  
13 M2 Ara-C, dauno, amsa, etop, mito CR  
14 M2 Ara-C, ida, amsa, asct CR 
15 M4 Ara-C, ida, amsa, etop, mito CR 
16 M2 Ara-C, dauno, amsa, etop, mito PR 
17 M2 Ara-C, etop, mito CR  
18 M4 Ara-C, dauno, asct CR  
19 M2 Ara-C, dauno, amsa, asct CR 
20 M0 Ara-C, dauno, amsa, asct PR 

Ida indicates idarubicin; amsa, amsacrine; asct, autologous stem cell transplantation, including conditioning regimen with busulfan and cyclophosphamide; etop, etoposide; mito, mitoxantrone; dauno, daunorubicin; CR, complete remission; PR, partial remission; and NR, no response.

BCRP protein expression in patient samples.

To study whether BCRP is expressed at the protein level in AML cells and whether it is up-regulated at relapse or refractory disease, we determined BCRP protein expression in the AML patient samples. In 38 of the 40 AML samples, a sufficient cell number was obtained to measure BCRP protein expression. The BXP-34/IgG1 ratio varied between 0.8 and 2.7 (1.6 ± 0.5, mean ± SD, n = 38) in the group of AML patients. The BXP-34/IgG1 ratios in all AML samples were lower than the observed ratios in the MCF-7 (9.8 ± 6.8) cell line. BCRP expression in the patient samples could be measured more sensitively with the BXP-21 monoclonal antibody; BXP-21/IgG2a ratios varied between 1.1 and 14.5 (4.9 ± 3.0, n = 38) in the whole group of AML samples versus ratios of 6.5 ± 2.4 in the MCF-7 and 11.3 ± 0.6 in the MCF-7 MR cell lines; 27 (73%) of the 37 samples showed BXP-21 levels below the level of the MCF-7 cell line, 9 (24%) patient samples showed BXP-21 levels between the MCF-7 and MCF-7 MR cell lines, and 1 (3%) patient sample showed a higher BXP-21/IgG2a ratio than the MCF-7 MR cell line. A correlation between BXP-34/IgG1 and BXP-21/IgG2a ratios was observed (r = 0.48, P = .003, n = 37) in the whole group of AML samples. The normal bone marrow mononuclear cells showed a BXP-34/IgG1 ratio of 1.7 ± 0.2 (n = 3), which was not significantly different from the patient samples, and a BXP-21/IgG2a ratio of 1.3 ± 0.1 (n = 3), which was lower than the patient samples (P < .05). Because the measurement of BCRP expression with BXP-21 appeared to be more sensitive than BXP-34 in the AML samples, these data were used in the following statistical analyses.

Although the overall expression of BCRP protein was slightly higher at relapse versus primary samples, no significant up-regulation of BCRP protein expression was observed at relapse (5.3 ± 3.3, n = 19) versus primary disease (4.4 ± 2.7, P = .4, n = 18). However, increases or decreases of BCRP protein expression at relapse could be observed in individual cases; BCRP expression was higher in 6 relapses and lower in 7 relapsed cases, whereas in 5 cases BCRP expression had not changed at relapse. Table4 shows the data of the individual patient samples.

Table 4.

BCRP protein expression and mitoxantrone accumulation in the individual AML patients

Patient no. BXP-21/IgG2a ratio Mitoxantrone, MFI 
Primary Relapse Primary Relapse 
1.57 1.70 761 302 
4.44 6.56 139 120  
1.97 6.98 445 115 
4.83 4.91 111 107  
2.74 2.15 270 419 
4.57 7.51 70 44  
2.85 1.98 165 177 
1.82 4.05 390 399  
8.77 9.24 96 89 
10 10.06 9.00 155 144 
11 3.88 2.94 179 202 
12 3.93 4.29 238 322 
13 4.42 3.52 488 269 
14 8.29 14.49 222 275 
15 1.08 1.25 422 292 
16 3.33 7.96 354 184 
17 nd nd 202 211 
18 2.34 4.63 109 115 
19 7.68 2.85 102 126 
20 5.88 5.14 127 107 
Patient no. BXP-21/IgG2a ratio Mitoxantrone, MFI 
Primary Relapse Primary Relapse 
1.57 1.70 761 302 
4.44 6.56 139 120  
1.97 6.98 445 115 
4.83 4.91 111 107  
2.74 2.15 270 419 
4.57 7.51 70 44  
2.85 1.98 165 177 
1.82 4.05 390 399  
8.77 9.24 96 89 
10 10.06 9.00 155 144 
11 3.88 2.94 179 202 
12 3.93 4.29 238 322 
13 4.42 3.52 488 269 
14 8.29 14.49 222 275 
15 1.08 1.25 422 292 
16 3.33 7.96 354 184 
17 nd nd 202 211 
18 2.34 4.63 109 115 
19 7.68 2.85 102 126 
20 5.88 5.14 127 107 

The results are given of BXP-21/IgG2a ratios and mitoxantrone accumulations of the individual patients at diagnosis of the AML and at relapse.

No effect of the particular chemotherapeutic agent used could be observed on the BCRP protein expression at relapse or refractory disease of the patients treated with daunorubicin (4.3 ± 2.1, n = 6), idarubicin (5.7 ± 3.8, n = 13), or mitoxantrone plus etoposide (4.6 ± 2.8, n = 11) versus de novo AML (4.4 ± 2.0, P = .75; 4.1 ± 3.0,P = .09; and 3.6 ± 2.6, P = .76; respectively).

The expression of BCRP protein correlated with the functional expression of MRP, as determined in a previous study21(r = 0.43, P = .007, n = 38), but not with the functional expression of P-gp (P = .12).

Mitoxantrone accumulation in patient samples.

The next purpose was to study whether a high BCRP protein expression was reflected by a high BCRP activity, resulting in a low mitoxantrone accumulation, and whether the BCRP activity was increased in relapsed or refractory AML. Therefore, the intracellular accumulation of mitoxantrone was determined after 60 minutes of exposure (10 μM) in the absence and presence of the BCRP inhibitor FTC (10 μM). In addition, the capacity of P-gp and MRP to extrude mitoxantrone was measured by incubating with mitoxantrone plus the inhibitors PSC833 (2 μg/mL) and MK-571 (20 μM). Furthermore, combinations of the 3 inhibitors were added to check whether BCRP, P-gp, and MRP showed an additive effect in extruding mitoxantrone. The intracellular accumulation of mitoxantrone in the absence of the inhibitors varied widely between the whole group of AML samples (mean 227 ± 146 MFI, range 44-761, n = 40), whereas the normal bone marrow samples showed a higher mitoxantrone accumulation of 695 ± 153 MFI, (P < .05, n = 3). In 17 patient samples the mitoxantrone accumulation was higher than the mitoxantrone level of the MCF-7 cell line (211 ± 15 MFI); in 20 patient samples the mitoxantrone level was between the levels of the MCF-7 and MCF-7 MR (93 ± 36 MFI) cell lines, and in 3 patient samples the mitoxantrone accumulation was even lower than the MCF-7 MR cell line.

The mitoxantrone accumulation in the whole group of relapsed samples was slightly lower (201 ± 107 MFI, n = 20) versus the whole group of primary samples (252 ± 176 MFI, n = 20), but the difference was not significant (P = .39). In 5 individual patient cases (patient nos. 1, 3, 13, 15, and 16) the accumulation of mitoxantrone was lower at relapse versus de novo AML, in 3 relapses (nos. 5, 12, and 14) the mitoxantrone accumulation was higher than in the primary samples, and in 12 cases no major differences (< 50 MFI) were found in mitoxantrone level between primary and relapsed or refractory samples (individual cases are shown in Table 4).

A strong correlation was observed between the intracellular mitoxantrone level and the expression of BCRP protein as measured with BXP-34 and BXP-21; patients with the highest expression of BCRP protein showed the lowest level of mitoxantrone accumulation (r = −0.37, P = .025 for BXP-34 andr = −0.58, P < .001 for BXP-21, n = 38) (Figure 2). In addition, a correlation existed between the capacity to extrude mitoxantrone and the functional activities of P-gp (r = −0.56, P < .001, n = 40) and MRP (r = −0.58, P < .001, n = 40), which were determined in these samples in the previous study,21 indicating that P-gp and MRP also extrude mitoxantrone in these patient samples. When the capacity of P-gp to extrude mitoxantrone was inhibited by PSC833, the correlation between high BCRP protein expression and low mitoxantrone accumulation remained present (r = −0.33, P = .048 for BXP-34 andr = −0.56, P < .001 for BXP-21, n = 38). In addition, when the capacity of MRP to extrude mitoxantrone was inhibited by MK-571, the correlation between high BCRP protein expression and low mitoxantrone accumulation was maintained (r = −0.37, P = .023 for BXP-34 andr = −0.48, P = .003 for BXP-21, n = 38). When both P-gp and MRP activity were inhibited using the combination of PSC833 and MK-571, which could be performed in 18 patient samples, the correlation between mitoxantrone accumulation and BCRP expression, as measured with BXP-34, was even stronger (r =  −0.57,P = .018, n = 18). However, this correlation was not observed between mitoxantrone accumulation and BXP-21 in this small group of patient samples (r = −0.36,P = .14, n = 18).

Fig. 2.

Correlation between BCRP protein expression and mitoxantrone accumulation in AML patient samples.

BCRP expression was measured with the BXP-21 monoclonal antibody, and mitoxantrone accumulation was measured after 60 minutes of incubation with mitoxantrone (10 μM).

Fig. 2.

Correlation between BCRP protein expression and mitoxantrone accumulation in AML patient samples.

BCRP expression was measured with the BXP-21 monoclonal antibody, and mitoxantrone accumulation was measured after 60 minutes of incubation with mitoxantrone (10 μM).

The addition of the BCRP inhibitor FTC (10 μM) showed a small increase of the mitoxantrone accumulation in the patient samples (11 ± 29 MFI). The addition of PSC833 alone showed an increase of 54 ± 56 MFI, and the addition of MK-571 alone showed an increase of 32 ± 55 MFI. The addition of FTC to the combination of PSC833 and MK-571 caused an increase of the shift from 80 ± 64 MFI to 98 ± 69 MFI (n = 18), which indicates that BCRP has a low capacity to transport mitoxantrone independently of P-gp and MRP.

Immunophenotype.

Because it has been described that the expression of P-gp and MRP are correlated with the expression of CD34,31 and with the early maturation stage, as determined by immune phenotyping,21 we were interested if BCRP protein was also especially expressed at the early stage in AML cells. Therefore, we determined the percentages of the immature (CD34+/CD38 and CD34+/CD33) and more mature (CD34+/CD33+ and CD34/CD33+) subclasses of cells. The percentages of cells in the immunophenotypic subclasses were correlated with BCRP protein expression and mitoxantrone accumulation. The results are shown in Table 5. Indeed, a high expression of BCRP protein, as measured with BXP-21, was correlated with a high percentage of CD34+ cells in the whole group of AML patients (r = 0.54, P = .001, n = 38), which was predominantly due to a correlation between the percentage of the immature CD34+/CD33 subclass of the CD34+ cells and BCRP expression (r = 0.40,P = .02, n = 38). A low BCRP protein expression was observed in the cases with a high percentage of the more mature CD34/CD33+ blasts (r = −0.56,P < .001, n = 38).

Table 5.

Correlations between immune phenotype, BCRP expression, and mitoxantrone accumulation

graphic
 
graphic
 

The results represent correlation coefficients between the percentages of the different immune phenotypic subclasses and BCRP protein expression, expressed as BXP-21/IgG2a ratios, and mitoxantrone accumulation, expressed as MFI. P values are presented in parentheses.

F5-150

Significant correlations.

These results were underlined by a correlation between a low mitoxantrone accumulation and a high percentage of CD34+cells (r = −0.35, P = .03, n = 40), especially the immature CD34+/CD38 subset of cells (r = −0.60, P < .001, n = 38). In addition, a correlation was found between a high mitoxantrone accumulation and a high percentage of CD33+ blasts (r = 0.45, P = .005, n = 38), which mainly consisted of the mature CD34/CD33+ cells (r = 0.61, P = .001, n = 38). Furthermore, these correlations became even stronger when the capacity of P-gp and MRP to extrude mitoxantrone was inhibited with PSC833 and MK-571 (Table5), indicating that the correlation between BCRP activity and immature immunophenotype is stronger than the correlations of P-gp and MRP activities with immature immunophenotype.

In summary, a relatively high BCRP activity and protein expression were observed in AML cells with an immature immunophenotype, whereas a low BCRP activity and protein expression were found in AML cells with a mature immunophenotype.

In addition, increases and decreases in the percentages of the immunophenotypic subclasses at relapse/refractory disease versus primary AML were analyzed (Table 6). The mean percentage of CD34+ cells in the whole group of AML samples was 54.1% ± 32.9% (n = 40). The percentage of CD34+ cells at relapse (61.7% ± 31.3%, n = 20) was increased versus de novo AML (46.5% ± 33.6%, P = .03, n = 20). The increase in CD34+ population was predominantly caused by an increase of the more mature CD34+/CD33+ subpopulation at relapse (43.1% ± 33.5%, n = 19) versus diagnosis (22.7% ± 24.3%,P = .004, n = 15). No difference was observed between primary and relapsed samples in the percentages of the other subclasses of cells.

Table 6.

Phenotype of the AML blasts at diagnosis and at relapse or refractory disease

graphic
 
graphic
 

nd indicates not done.

F6-150

Significant difference between primary and relapsed/refractory samples.

Treatment with idarubicin or daunorubicin was not correlated with an increase of any of the specific subsets of AML cells. The AML patients who were not treated with mitoxantrone and etoposide but received an autograft transplantation showed a higher percentage of CD34+ cells (78.0% ± 22.3%) and of CD33+cells (60.8% ± 36.8%) at relapse as compared with the primary AML (55.8% ± 31.7%, P = .01, n = 9 for CD34 and 43.6% ± 35.6%, P = .04, n = 9 for CD33). This was predominantly caused by an increase in the CD34+/CD33+ subfraction at relapse (53.8% ± 35.8%) versus the primary AML (26.6% ± 27.5%,P = .01, n = 9).

Discussion

The present study demonstrates for the first time that BCRP protein expression can be analyzed by flow cytometry in AML samples. Thus far, BCRP expression in AML samples has been studied at the mRNA level20,32 or at protein level with immunohistochemistry.22 In the latter study using the monoclonal antibody BXP-34, we observed no BCRP protein expression in the MCF-7 cell line and in AML samples.22 The flow cytometric assay presented in the current study appeared to be more sensitive, because distinct levels of BCRP expression were observed in the MCF-7 cell line and the AML patient samples.

In the flow cytometric assay BXP-21 was more sensitive than BXP-34 for patient samples, whereas in the cell lines the reverse was noticed. This difference was predominantly due to high values of the IgG2a isotype control in the cell lines. A low correlation was observed between BXP-21 and BXP-34 expression (r = 0.48), which can be explained by the low and therefore insensitive BXP-34 expression levels.

BCRP protein expression correlated strongly with mitoxantrone accumulation, with patients with relatively high BCRP protein expression showing a low mitoxantrone accumulation. When P-gp and MRP activity were inhibited with PSC833 or MK-571 alone, the correlation with BCRP expression, as determined with the monoclonal antibodies BXP-34 and BXP-21, remained. When both P-gp and MRP activity were inhibited simultaneously with the combination of PSC833 and MK-571, the correlation between mitoxantrone accumulation and BCRP expression remained in the case of the BXP-34 monoclonal antibody. The specific epitopes of the BCRP protein that are recognized by BXP-34 and BXP-21, respectively, have not yet been identified. In the present study as well as in an additional study (unpublished data), we observed a better correlation between BCRP activity and expression as measured with BXP-34 versus the BXP-21 monoclonal antibody in clinical samples. We therefore hypothesize that BXP-21, although it specifically binds to BCRP and shows no cross-reactivity with other proteins,22might recognize the BCRP protein only in a not functionally active state in these clinical samples.

The reversing substance FTC appeared to increase effectively the mitoxantrone accumulation in the cell lines. A small increase of the mitoxantrone accumulation was observed upon addition of FTC in the patient samples. Moreover, the addition of FTC to the combination of PSC833 and MK-571 caused an additional increase in mitoxantrone accumulation, which confirms that BCRP transports mitoxantrone in addition to P-gp and MRP. PSC833 had a more distinct effect on the accumulation of mitoxantrone than MK-571 and FTC. Therefore, P-gp seems to play the most important role in extruding mitoxantrone in the AML patient samples. These findings emphasize that the resistance mediated by drug efflux pumps is a complex phenomenon and might provide a plausible explanation for the disappointing results of the clinical use of a specific inhibitor of only one transporter protein.33,34 Moreover, it has been demonstrated that the inhibition or absence of one transporter protein might force or induce the activity of additional transporters,35,36 which may further limit the effectivity of the selective inhibition of transporter proteins.

For P-gp and MRP1 it has been observed that selected cell lines acquire much higher levels of P-gp and MRP1 expression and function than clinical samples.30 The P-gp and MRP1 expression and function in the clinical AML samples appeared, however, to correlate with drug resistance4,6,7; BCRP expression might follow the same pattern.

BCRP protein expression was not consistently up-regulated at relapse or refractory disease. In some cases BCRP expression was higher and in other cases lower at relapse, which was related to the immunophenotype of the AML cells. A strong positive correlation was observed between BCRP protein expression and immature phenotype, reflected by CD34 positivity, whereas a strong negative correlation was found between BCRP protein expression and a more mature phenotype (CD34/CD33+). Interestingly, the correlation between BCRP expression and maturation stage has been described recently for normal hematopoietic cells. BCRP expression has been observed especially in the most primitive and quiescent hematopoietic progenitors,23-25 as has also been found for P-gp and MRP,21,31 suggesting a role for the transporter proteins in maintaining a primitive phenotype in hematopoietic cells, as has been previously described for an ABC transporter in Dictyosteliumcells.37 Alternatively, it has been described that P-gp plays a role in protecting cells from apoptosis,38,39which also might be the case in stem cells or primitive AML cells with high expressions of P-gp as well as BCRP.

A recently presented study reported an up-regulation of BCRP mRNA expression at relapse in a group of 20 paired samples of primary and relapsed or refractory AML in which a 1.7-fold increase of mRNA expression was observed.32 The present study did not confirm these data with BCRP protein expression. Because both studies present the results of a small and selected group of 20 paired AML samples, this might explain the discrepant results.

An increased percentage of CD34+ cells was detected at relapse or refractory disease versus de novo AML, mainly consisting of the CD34+/CD33+ subpopulation of cells, which is consistent with findings in other studies describing increased CD34+ 40 and CD33+ 40,41 populations at relapse in AML.

In summary, the present study shows that BCRP protein is expressed in AML blasts at variable levels. Furthermore, BCRP in AML blasts, although at a low level, seems capable of actively transporting mitoxantrone, a substrate used in the treatment of AML, and therefore appears to be an additional transporter in AML. BCRP expression and reversal of BCRP-mediated transport should be studied in a larger group of AML patients in the future to determine the effect of BCRP expression and activity on clinical outcome. BCRP expression, as also observed for P-gp and MRP, was not consistently up-regulated in all relapsed/refractory AML cases but was up-regulated in some cases and decreased in other cases and appeared to be correlated with immature immunophenotype.

Supported by a grant of the Foundation of Pediatric Oncology Groningen (SKOG 99-01).

Submitted August 7, 2001; accepted January 9, 2002.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

References

References
1
Simon
M
Schindler
M
Cell biological mechanisms of multidrug resistance in tumor.
Proc Natl Acad Sci U S A.
91
1994
3497
3504
2
Cole
SPC
Bhardwaj
G
Gerlach
JH
et al. 
Overexpression of a novel transporter gene in a multidrug resistant human lung cancer cell line.
Science.
258
1992
1650
1654
3
Müller
M
Meyer
C
Zaman
GJ
et al. 
Overexpression of the gene encoding the multidrug resistance-associated protein results in increased ATP-dependent glutathione S-conjugate transport.
Proc Natl Acad Sci U S A.
91
1994
13033
13037
4
Legrand
O
Simonin
G
Beauchamp-Nicoud
A
Zittoun
R
Marie
J-P
Simultaneous activity of MRP1 and Pgp is correlated with in vitro resistance to daunorubicin and with in vivo resistance in adult acute myeloid leukemia.
Blood.
94
1999
1046
1056
5
Leith
CP
Kopecky
KJ
Chen
IM
et al. 
Frequency and clinical significance of the expression of the multidrug resistance proteins MDR1/P-glycoprotein, MRP1, and LRP in acute myeloid leukemia: a Southwest Oncology Group Study.
Blood.
94
1999
1086
1099
6
Van der Kolk
DM
de Vries
EGE
van Putten
WLJ
et al. 
P-gp and MRP activities in relation to treatment outcome in acute myeloid leukemia.
Clin Cancer Res.
6
2000
3205
3214
7
Legrand
O
Perrot
JY
Sinonin
G
Baudard
M
Marie
JP
JC-1: a very sensitive fluorescent probe to test Pgp activity in adult acute myeloid leukemia.
Blood.
97
2001
502
508
8
Doyle
LA
Yang
W
Abruzzo
LV
et al. 
A multidrug resistance transporter from human MCF-7 breast cancer cells.
Proc Natl Acad Sci U S A.
95
1998
15665
15670
9
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.
58
1998
5337
5339
10
Ross
DD
Yang
WD
Abruzzo
LV
et al. 
A typical multidrug resistance: breast cancer resistance protein messenger RNA expression in mitoxantrone-selected cell lines.
J Natl Cancer Inst.
91
1999
429
433
11
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.
63
1991
923
929
12
Lee
YJ
Galoforo
SS
Berns
CM
Tong
WP
Kim
HR
Corry
PM
Glucose deprivation-induced cytotoxicity in drug resistant human breast carcinoma MCF-7/ADR cells: role of c-myc and bcl-2 in apoptotic cell death.
J Cell Sci.
110
1997
681
686
13
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.
59
1999
4559
4563
14
Litman
T
Brangi
M
Hudson
E
et al. 
The multidrug-resistant phenotype associated with overexpression of the new ABC half-transporter, MXR (ABCG2).
J Cell Sci.
113
2000
2011
2021
15
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.
58
1998
5850
5858
16
Schiller
G
Gajewski
J
Territo
M
Long-term outcome of high-dose cytarabine-based consolidation chemotherapy for adults with acute myelogenous leukemia.
Blood.
80
1992
2977
2982
17
Cassileth
PA
Harrington
DP
Appelbaum
FR
et al. 
Chemotherapy compared with autologous or allogeneic bone marrow transplantation in the management of acute myeloid leukemia in first remission.
N Engl J Med.
339
1998
1649
1656
18
Godwin
JE
Kopecky
KJ
Head
DR
A double-blind placebo-controlled trial of granulocyte colony-stimulating factor in elderly patients with previously untreated acute myeloid leukemia: a Southwest Oncology Group Study (9031).
Blood.
15
1998
3607
3615
19
Löwenberg
B
Van Putten
WLJ
Verdonck
LF
Autologous bone marrow transplantation in acute myeloid leukemia in first remission: results of a Dutch prospective study.
J Clin Oncol.
8
1990
287
294
20
Ross
DD
Karp
JE
Chen
TT
Doyle
LA
Expression of breast cancer resistance protein in blast cells from patients with acute leukemia.
Blood.
96
2000
365
368
21
van der Kolk
DM
de Vries
EGE
Noordhoek
L
et al. 
Activity and expression of the multidrug resistance proteins P-glycoprotein, MRP1, MRP2, MRP3 and MRP5 in de novo and relapse acute myeloid leukemia.
Leukemia.
15
2001
1544
1553
22
Scheffer
GL
Maliepaard
M
Pijnenborg
ACLM
et al. 
BCRP is localized at the plasma membrane in mitoxantrone- and topotecan-resistant cell lines.
Cancer Res.
60
2000
2589
2593
23
Maliepaard
M
Scheffer
GL
Faneyte
IF
et al. 
Subcellular localization and distribution of the breast cancer resistance protein transporter in normal human tissues.
Cancer Res.
61
2001
3458
3464
24
Scharenberg
CW
Harkey
MA
Torok-Storb
BJ
The ABCG2 transporter is an efficient Hoechst 33342 efflux pump and is preferentially expressed by immature human hematopoietic progenitors.
Blood.
99
2002
507
512
25
Zhou
S
Schuetz
JD
Bunting
KD
et al. 
The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype.
Nat Med.
7
2001
1028
1034
26
Löwenberg
B
Suciu
S
Archimbaud
E
et al. 
Mitoxantrone versus daunorubicin in induction-consolidation chemotherapy: the value of low-dose cytarabine for maintenance of remission, and an assessment of prognostic factors in acute myeloid leukemia in the elderly: final report. European Organization for the Research and Treatment of Cancer and the Dutch-Belgian Hemato-Oncology Cooperative Hovon Group.
J Clin Oncol.
15
1998
872
881
27
Vellenga
E
van Putten
WLJ
Boogaerts
MA
et al. 
Peripheral blood stem cell transplantation as an alternative to autologous marrow transplantation in the treatment of acute myeloid leukemia.
Bone Marrow Transplant.
23
1999
1279
1289
28
Jones
TR
Zamboni
R
Belley
M
et al. 
Pharmacology of L-660,711 (MK-571): a novel potent and selective leukotriene D4 receptor antagonist.
Can J Physiol Pharmacol.
67
1989
17
18
29
Robey
RW
Medina-Pérez
WY
Nishiyama
K
et al. 
Overexpression of the ATP-binding cassette half-transporter, ABCG2 (MXR/BCRP/ABCP1), in flavopiridol-resistant human breast cancer cells.
Clin Cancer Res.
7
2001
145
152
30
van der Kolk
DM
de Vries
EGE
Koning
JA
van den Berg
E
Muller
M
Vellenga
E
Activity and expression of the multidrug resistance proteins MRP1 and MRP2 in acute myeloid leukemia cells, tumor cell lines, and normal hematopoietic CD34+ peripheral blood cells.
Clin Cancer Res.
4
1998
1727
1736
31
Te Boekhorst
PAW
de Leeuw
K
Schoester
M
et al. 
Predominance of functional multidrug resistance (MDR-1) phenotype in CD34+ acute myeloid leukemia cells.
Blood.
82
1993
3157
3162
32
Van den Heuvel-Eibrink
MM
Wiemer
EAC
Prins
A
Meijerink
JPP
et al. 
Increased expression of the novel drug resistance gene BCRP, but not of MDR1, LRP/MVP, and MRP1 in relapsed/refractory AML using real time quantitative RT-PCR (Taqman analysis) [abstract].
Leukemia.
15
2001
487
33
Greenberg
P
Advani
R
Tallman
M
et al. 
Treatment of refractory/relapsed AML with PSC833 plus mitoxantrone, etoposide, cytarabine (PSC-MEC) vs MEC: randomized phase III trial (E2995) [abstract].
Blood
94
1999
383a
34
Baer
MR
George
SL
Dodge
RK
et al. 
Phase III study of PSC-833 modulation of multidrug resistance (MDR) in previously untreated acute myeloid leukemia (AML) patients (pts) ≥ 60 years (CALBG9720) [abstract].
Blood
94
1999
383a
35
Van der Kolk
DM
Vellenga
E
van der Veen
AY
et al. 
Deletion of the multidrug resistance protein MRP1 gene in acute myeloid leukemia: the impact on MRP activity.
Blood.
95
2000
3514
3519
36
Konig
J
Rost
D
Cui
Y
Keppler
D
Characterization of the human multidrug resistance protein isoform MRP3 localized to the basolateral hepatocyte membrane.
Hepatology.
29
1999
1156
1163
37
Good
RJ
Kuspa
A
Evidence that a cell-type-specific efflux pump regulates cell differentiation in Dictyostelium.
Dev Biol.
220
2000
53
61
38
Smyth
MJ
Krasovskis
E
Sutton
VR
Johnstone
RW
The drug efflux protein, P-glycoprotein, additionally protects drug-resistant tumor cells from multiple forms of caspase-dependent apoptosis.
Proc Natl Acad Sci U S A.
95
1998
7024
7029
39
Johnstone
RW
Cretney
E
Smyth
MJ
P-glycoprotein protects leukemia cells against caspase-dependent, but not caspase-independent, cell death.
Blood.
93
1999
1075
1085
40
Baer
MR
Stewart
CC
Dodge
RK
et al. 
High frequency of immunophenotype changes in acute myeloid leukemia at relapse: implications for residual disease detection (Cancer and Leukemia Group B Study 8361).
Blood.
97
2001
3574
3580
41
Thomas
X
Campos
L
Archimbaud
E
et al. 
Surface marker expression in acute myeloid leukemia at first relapse.
Br J Haematol.
81
1992
40
44

Author notes

E. G. E. de Vries, Division of Medical Oncology, Dept of Internal Medicine, University Hospital Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands; e-mail:e.g.e.de.vries@int.azg.nl.