|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Prepublished online as a Blood First Edition Paper on July 25, 2002; DOI 10.1182/blood-2002-01-0271.
NEOPLASIA
From the Department of Hematology/Oncology, St Jude
Children's Research Hospital, Memphis, TN; and the Department of Blood
and Marrow Transplantation, Section of Molecular Hematology and
Therapy, M. D. Anderson Cancer Center, Houston, TX.
Previous reports have suggested that the adenosine
triphosphate-binding cassette protein ABCG2 (breast cancer
resistance protein [BCRP], mitoxantrone resistance [MXR])
is associated with drug resistance in acute myeloid leukemia (AML). The
aims of this study were to determine the level of ABCG2 mRNA expression
necessary to produce drug resistance and to define the ABCG2 levels in
normal bone marrow (BM), peripheral blood (PB), cord blood (CB), and adult AML blast cell populations. First, using transduced clonal cell
lines expressing varying levels of ABCG2, we found that ABCG2 expression conferred resistance to mitoxantrone and topotecan, but not
to idarubicin. Next, we developed a real-time reverse transcription-polymerase chain reaction assay for measuring
ABCG2 mRNA expression levels in clinical samples. Normal BM and PB
contained low levels of ABCG2 mRNA, while higher levels were measured
in CB mononuclear cells, CD34+, and Ac133+
populations, consistent with the known stem cell enrichment in these
populations. Next, we studied the ABCG2 mRNA levels in 40 specimens
from newly diagnosed adult AML patients. Only 7% of these samples
contained ABCG2 mRNA levels within the range of our drug-resistant
clone, although another 78% were higher than normal blood and bone
marrow. Flow cytometry revealed very small subpopulations of
ABCG2-expressing cells in the cases we examined. Our data suggest that
high levels of ABCG2 mRNA expression in adult AML blast specimens are
relatively uncommon and that ABCG2 expression may be limited to a small
cell subpopulation in some cases.
(Blood. 2002;100:4594-4601) Drug resistance is a frequently encountered
phenomenon contributing to treatment failure in acute leukemia.
Several members of the adenosine triphosphate-binding cassette
(ABC) family of transmembrane transporters have been associated with
drug resistance in acute myeloid leukemia (AML), including
P-glycoprotein,1,2 encoded by the multidrug resistance
gene, MDR1, and the multidrug resistance protein,
MRP1.3 Several studies have suggested that other
transporters may be responsible for the multidrug resistance phenotype
in some AML cases. Leith et al showed that 18% of adult AML specimens
had drug efflux activity, which was not associated with MDR1 or MRP1
expression, suggesting the presence of a previously unidentified
transporter.4
The transporter ABCG2, also designated BCRP and MXR, was recently
described as a cause of the multidrug resistance
phenotype.5 ABCG2 mRNA expression was identified in
approximately one third of cases of adult AML by semiquantitative
reverse transcription-polymerase chain reaction
(RT-PCR).6 Another study detected ABCG2 protein expression
in 27% of cases by flow cytometry.7 However, it is
unclear if the ABCG2 mRNA expression measured in these samples was
physiologically significant or due to contamination from normal hematopoietic cells in low-expressing cases. Because ABCG2 expression has subsequently been demonstrated in several types of normal hematopoietic cells, including erythroid progenitors, natural killer
cells, and primitive stem cells,8,9 expression in AML
blast samples could be due to contamination from normal hematopoietic cells.
The substrate profile of ABCG2 includes the antineoplastic drugs
mitoxantrone and topotecan.10 Mitoxantrone is commonly used in AML treatment, suggesting that ABCG2 could play an important role in drug resistance in this disease. Idarubicin is another anthracycline drug that is sometimes used in the treatment of AML, and
it has not been known whether ABCG2 expression can confer resistance to idarubicin.
ABCG2 is known to efflux the fluorescent dye, Hoescht 33342, but not
rhodamine 123.8,11 This dye efflux activity can be used to
identify hematopoietic stem cells using the "side population" (SP)
assay,12,13 and previous studies have identified ABCG2 expression as a molecular determinant of the SP
phenotype.8,9 This finding may be important in AML for
several reasons. First, several investigators have found the frequent
presence of SP cells in bone marrow from AML
patients.14,15 While in both studies the SP cells were
usually leukemic, Feuring-Buske et al demonstrated that specimens from
most AML patients contained both leukemic and nonleukemic SP
cells.15 Interestingly, these SP cells frequently did not
express MDR1 and were capable of multilineage NOD/SCID engraftment.
These studies provide further evidence that a transporter such as ABCG2
may be expressed in stem cells of both normal and leukemic origin.
To assess ABCG2 expression in AML patient samples, we developed clonal
human AML cell lines by transduction with a retroviral vector
containing the ABCG2 cDNA. Using these clonal cell lines, we identified
a low-expressing clone, referred to as clone 3.3, that has readily
detectable levels of ABCG2 mRNA by real-time RT-PCR analysis but very
low levels of protein expression and no drug resistance. Another clone,
6.2 with ABCG2 mRNA expression much higher than clone 3.3, demonstrated
relatively high levels of protein expression by monoclonal antibody
labeling and mitoxantrone efflux assays. Resistance to several relevant
cytotoxic drugs was then measured in these cell lines to develop a
correlation between ABCG2 mRNA levels and functional drug resistance.
The mRNA levels in these reference cell lines were also compared to those seen in normal hematopoietic cell populations and in 40 samples
from adult AML patients. In several cases, blast cell populations were
studied for ABCG2 protein expression using a monoclonal antibody we
have recently generated. These expression and functional studies
further clarify the role of ABCG2 expression in AML.
Preparation of ABCG2-transduced clonal AML cell lines
Drug efflux assays
SP analyses and antibody-mediated inhibition of efflux Thirty micrograms of 5D3 anti-ABCG2 antibody8 (prepared by our laboratory) or isotype control (mouse IgG2b, Caltag, Burlingame, CA) were added per 1 million cells in a total volume of 100 µL serum-free DMEM and then incubated on ice for 30 minutes. After this incubation step, Hoechst 33342 (Sigma) was diluted in DMEM with 2% fetal calf serum (FCS) plus 1 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) and added to cells for a final concentration of 5 µg/mL and a final volume of 1.0 mL per million cells. After incubation at 37°C for 90 minutes, the cells were centrifuged at 1200 rpm for 5 minutes and resuspended in cold Hanks balanced salt solution with 2% FCS + 1 mM HEPES. Cells were kept on ice until flow analysis for SP cells was performed as previously described.13Normal hematopoietic cell assays Under approved institutional protocols, 4 samples each of bone marrow, peripheral blood, and umbilical cord blood were obtained from healthy volunteer donors. Peripheral blood stem cells (PBSCs) were obtained from healthy volunteer donors after mobilization using filgrastim (G-CSF; Amgen, Thousand Oaks, CA) at a dose of 480 µg/day for 4 days. PBSC collection was performed by double-volume apheresis, using the Cobe Spectra LRS-Turbo7 (Cobe Laboratories, Lakewood, CO).Mononuclear cells were obtained using ficoll separation (Histopaque-1077, Sigma) according to the manufacturer's recommendations. Following recovery of mononuclear cells, further purification was performed by resuspending in red cell lysis buffer (8.3 g/L ammonium chloride with potassium bicarbonate 1.0 g/L) for 15 minutes, centrifuging, then resuspending in PBS. RNA was extracted from the resulting cell pellet using RNA STAT-60 (Tel-Test, Friendswood, TX) according to the manufacturer's recommendations. CD34 and Ac133 cell enrichments were performed on mobilized peripheral blood leukapheresis products using the CliniMACS instrument (CS2 CE/UL, Miltenyi Biotec, Gladbach, Germany) and CD34 and Ac133 CliniMACS reagents and using the manufacturer's protocols. RNA was extracted using RNA STAT-60. AML blast samples Bone marrow or peripheral blood leukapheresis samples were obtained from 40 patients with newly diagnosed AML at the M. D. Anderson Cancer Center in Houston, TX. Leukemic cells were obtained from bone marrow in 32 cases, and from peripheral blood leukapheresis in the other 8 cases. The samples contained between 30% and 98% blasts. 5 × 106 to 2 × 107 cells were frozen in medium containing 90% FCS and 10% DMEM. Cells were warmed and immediately centrifuged at 1500 rpm for 3 minutes. RNA was extracted from the resulting cell pellet using the RNA STAT-60.Quantitative real-time RT-PCR Levels of ABCG2 and -actin expression were determined for
each sample using quantitative real-time RT-PCR, in separate reactions. Briefly, to each well of a 96-well plate were added 100 ng RNA, 12.5 µL Universal 2 × PCR Master Mix (Applied Biosystems, Foster City,
CA), 0.625 µL 40 × RT Buffer (Applied Biosystems), primers (300 nM each), probe (100 nM), and diethyl pyrocarbonate-treated water, to a total volume of 25 µL per well. For ABCG2, the
sequence of the forward primer was 5'-TGCAACATGTACTGGCGAAGA-3'; the
sequence of the reverse primer was 5'-TCTTCCACAAGCCCCAGG-3'; and the
sequence of the probe was
5'-fam-TATTTGGTAAAGCAGGGCATCGATCTCTCA-tamra-3'. For -actin, the
sequence of the forward primer was 5'-AGTACTCCGTGTGGATCGGC-3'; the
sequence of the reverse primer was 5'-GCTGATCCACATCTGCTGGA-3'; and the
sequence of the probe was 5'-vic-CCATCCTGGCCTCGCTGTCCA-tamra-3'. Thermal cycler conditions included an initial reverse transcription step at 48°C for 30 minutes, followed by incubation at 95°C for 10 minutes, then 40 cycles alternating between 95°C for 15 seconds and
60°C for 1 minute. Results were collected and analyzed using an ABI
Prism 7700 Sequence Detection System (Applied Biosystems).
Reaction data were expressed as cycle thresholds (Ct), which is the PCR
cycle number at which the fluorescent signal in each reaction reaches a
threshold above background. Samples were normalized for RNA loading by
subtracting the Ct of ABCG2 from the Ct of Northern analysis Northern analysis was performed as previously described.17 A full-length ABCG2 cDNA probe was prepared by restriction digest of the pHaABCG2 plasmid with the enzymes NcoI and SacII (Promega, Madison, WI). The probe was radiolabeled with dCTP32 using the NEBlot Kit according to the manufacturer's instructions (New England BioLabs, Beverly, MA) and hybridized at 42°C for 12 hours.ABCG2 expression was normalized to Flow cytometry analysis 1 × 106 cells were harvested from suspension culture (for cell lines) or thawed bone marrow or leukapheresis sample (for patient specimens) and washed twice with 5 mL PBSA (1% bovine serum albumin in PBS). Cells were labeled with 1 µg of anti-ABCG2 monoclonal phycoerythrin-conjugated antibody 5D3 (E-Bioscience, San Diego, CA) at room temperature for 30 minutes. Cells were washed twice with 5 mL PBSA, then resuspended in 500 µL PBSA and analyzed by flow cytometry. Propidium iodide (PI) was added, and all analyses were performed on PI-dull (live) cells. Results were compared to labeling with a PE-conjugated mouse IgG2b isotype control antibody (Caltag). Results were expressed as the ratio of mean fluorescent intensity for 5D3 to isotype for cell lines. For patient samples, results are expressed as percent positive cells by 5D3 labeling, and also as the ratio of mean fluorescent intensity of 5D3 compared to isotype.Drug sensitivity assays The parental OCI-AML3 cell line and ABCG2-transduced clones designated 3.3 and 6.2 were harvested and placed in 96-well plates at a density of 1 × 105 cells in 50 µL medium per well. For drug sensitivity testing, mitoxantrone (Novantrone, Immunex), topotecan (Hycamtin, GlaxoSmithKline, Research Triangle Park, NC), and idarubicin (Sigma) were each diluted in DMEM medium with 10% FCS to 2 × working concentrations. To achieve the indicated concentrations, 50 µL of each dilution was added to the plated cells. Cells were incubated with drug at 37°C in 5% CO2 for 96 hours.To assess the number of viable cells, a modified 3-(4,5-dimethyltriazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed. Briefly, 20 µL CellTiter 96 Aqueous solution (Promega) was added to each well and incubated at 37°C for 1 hour. Ultraviolet absorption was measured using a plate reader with a 492-nm filter, and absorbance was measured at 650 nm. Results were calculated by subtracting the background absorbance obtained with plain medium from the absorbance of each experimental sample. Percentage viability was determined by dividing the absorbance of each sample by the absorbance of untreated wells. Results are expressed as the means of 6 replicate determinations, relative to the amount of viability detected in the untreated wells. Statistical methods Statistics were calculated using GraphPad Prism, version 3.02 for Windows (GraphPad Software, San Diego, CA). Normality of results distribution was analyzed using the KS test. Normal hematopoietic cell populations were compared using the 2-tailed unpaired Student t test. Nonparametric data sets from leukemic patient samples were compared with the Mann-Whitney U test.
Characterization of ABCG2-expressing AML cell lines To develop cell line standards for ABCG2 mRNA expression, we prepared clonal AML cell lines based on the parental AML cell line, OCI-AML3.16 These cells were transduced with the HaABCG2 retroviral vector,8 and single transduced cells were isolated using a single-cell sorter. Numerous clones were expanded and screened for ABCG2-mediated mitoxantrone efflux activity. Based on their respective low and high levels of efflux activity, 2 individual clones, designated 3.3 and 6.2, were chosen for subsequent studies.First, the clones were analyzed by flow cytometry for their capacity to
efflux mitoxantrone. The parental OCI-AML3 cells showed no intrinsic
efflux capacity, clone 3.3 had a trace amount of efflux, and clone 6.2 displayed strong efflux activity (Figure 1A). The mean fluorescence intensities
for the cell lines were 2298 for OCI-AML3, 2300 for clone 3.3, and 252 for clone 6.2. The percentage of cells appearing as mitoxantrone dim
was 0.3% for parental OCI-AML3 cells, 21.7% for clone 3.3 cells, and
98.8% for clone 6.2 cells.
We analyzed the presence of ABCG2 surface protein by flow cytometry using an anti-ABCG2 monoclonal antibody, 5D3, which recognizes an external epitope on the plasma membrane of living cells.8 OCI-AML3 showed no significant labeling with the 5D3 antibody, clone 3.3 showed trace labeling, while clone 6.2 was strongly positive (Figure 1A). These results show that clone 6.2 expressed relatively high levels of ABCG2 protein, while clone 3.3 expressed much lower levels. The mean fluorescence intensity ratios of 5D3 to isotype were 1:1 for OCI-AML3, 1.6:1 for clone 3.3, and 32.7:1 for clone 6.2. The percentage of cells within the positive gate were 0.0% for parental AML3 cells, 1.4% for clone 3.3, and 99.5% for clone 6.2. To ensure that clone 3.3 did not contain aberrantly localized ABCG2 protein, immunofluorescence studies were performed on the AML3, 3.3, and 6.2 cell lines using the fluorescein isothiocyanate-conjugated BXP-21 anti-ABCG2 antibody.18 This revealed no detectable labeling in parental AML3, strong membrane labeling in clone 6.2, and a low level of membrane labeling in clone 3.3 with no evidence of aberrant subcellular localization (data not shown). To further test for ABCG2 function in these cell lines, we performed SP analyses by staining cells with Hoechst 33342 and using flow cytometry to quantify SP cell numbers.13 We and others have previously shown that enforced ABCG2 expression confers the SP phenotype to transduced cells.8,9 As expected, clone 6.2 showed a large increase in SP cells relative to the untransduced AML3 parental line, and clone 3.3 showed a much smaller increase in SP cells over the parental line (Figure 1B, top row). The 5D3 monoclonal antibody inhibits ABCG2 function, presumably by binding to an external epitope and preventing conformational changes in the transporter, and therefore provides a specific inhibitor assay for ABCG2 function. When cells were incubated with 5D3 prior to the SP analyses, both the 6.2 and 3.3 lines showed a marked decrease in SP cells (Figure 1B, bottom row). This result verifies the relative degrees of specific ABCG2 function in both the 6.2 and 3.3 cell lines. We also analyzed the capacity of clone 6.2 to efflux the anthracycline, idarubicin, a commonly used drug in AML induction chemotherapy. In contrast to the results seen with mitoxantrone, no significant efflux of idarubicin was detected (Figure 1C). To measure drug resistance of the 2 clones, we used a modified MTT assay, which measured cell viability following 96 hours of continuous drug incubation with mitoxantrone, topotecan, or idarubicin. We found that clone 6.2 was highly resistant to mitoxantrone and topotecan, but not to idarubicin (Figure 2A-C). In contrast, clone 3.3 showed no significant resistance to any of the agents. These results demonstrate that (1) the level of ABCG2 expression in clone 6.2 confers significant resistance to mitoxantrone and topotecan, (2) the low level of ABCG2 expression in clone 3.3 does not confer drug resistance in these assays, and (3) idarubicin is not a substrate for ABCG2 and can kill the ABCG2-expressing 6.2 cells. We have not yet determined if this idarubicin sensitivity can be generalized to other ABCG2-expressing cell lines. ABCG2 mRNA expression in AML clones To measure ABCG2 mRNA expression levels in the AML clones, Northern analysis of cellular RNA was performed. Clone 6.2 had relatively high levels of ABCG2 transcript, while clone 3.3 had much lower levels of ABCG2 mRNA (Figure 3). The parental OCI-AML3 cells had no detectable ABCG2 mRNA expression. Using-actin as an internal control to normalize for loading, the
level of ABCG2 mRNA in clone 6.2 was 1900% that of clone 3.3, and
parental OCI-AML 3 cells had 2.8% the level of clone 3.3.
To enable more sensitive measurements of ABCG2 mRNA expression levels
in clinical samples, we developed a quantitative real-time RT-PCR assay
using these cell line controls. The amplification range of the
real-time RT-PCR assay was demonstrated to be linear for both the ABCG2
and Expression in the 6.2 and 3.3 clonal cell lines was measured by
real-time RT-PCR (Figure 4) and compared
to results from Northern analysis. By real-time RT-PCR, untransduced
OCI-AML3 cells contained 0.2% of the ABCG2 level found in clone 3.3, versus 2.8% by Northern analysis. By real-time RT-PCR, clone 6.2 contained 2500% of the ABCG2 mRNA level found in clone 3.3, versus
1900% by Northern analysis. Thus, the PCR assay correlated fairly well
with the Northern blot analysis. The ABCG2 mRNA level seen in clone 3.3 defined a level of expression insufficient to confer drug resistance but readily detectable by the real-time RT-PCR assay.
ABCG2 mRNA expression in normal hematopoietic cell populations Expression of the murine ortholog of ABCG2 has been described previously in primitive stem cell populations from bone marrow, skeletal muscle, and embryonic stem cells of mice.8 Normal human hematopoietic populations also have been shown to express ABCG2 mRNA.9 Because leukemic bone marrow and peripheral blood specimens are likely to contain at least some residual normal cells with ABCG2 expression, we quantified this background level in order to interpret ABCG2 levels in leukemic samples. To define the ABCG2 mRNA expression in normal hematopoietic cells, we analyzed RNA from mononuclear cell preparations of human bone marrow (BM), peripheral blood (PB), and umbilical cord blood (CB) specimens by real-time RT-PCR. ABCG2 mRNA levels were relatively low in bone marrow (1.4% ± 0.8% that of clone 3.3), peripheral blood (1.2% ± 0.4%), and umbilical cord blood (6.4% ± 0.9%) populations (Figure 5A). These very low levels are consistent with our recent results showing that expression in murine hematopoietic tissues was relatively restricted to rare repopulating stem cells.8 ABCG2 mRNA expression in CB was significantly higher than levels in BM (P = .015) and PB (P = .002), consistent with the known higher stem cell frequency in cord blood.19
To determine the ABCG2 mRNA levels in human hematopoietic cell populations enriched for hematopoietic stem cells, we studied RNA samples from granulocyte colony-stimulating factor-mobilized peripheral blood stem cell (PBSC) populations magnetically immunopurified for either CD34 or Ac133 antigen expression (> 95% purity). The mean ABCG2 mRNA level of mobilized PB by real-time RT-PCR was 1.2% that of clone 3.3 (± 0.1%). Compared to unsorted PBSCs, CD34+ and Ac133+ populations contained significantly higher mean ABCG2 mRNA levels (Figure 5B), which were 15.6% ± 2.7% (P = .002), and 10.3% ± 3.6% (P = .015), respectively. These results are consistent with enrichment of primitive hematopoietic stem cells previously shown to be present in these populations.19,20 The low ABCG2 levels relative to clone 3.3 are consistent with the specificity of ABCG2 expression in hematopoietic stem cells and the overall low proportion of stem cells in these immunopurified populations. ABCG2 mRNA levels in adult AML specimens To determine the range of ABCG2 mRNA expression in blast cells from adults with AML, RNA samples from 40 AML bone marrow or leukapheresis specimens from previously untreated adult AML patients were analyzed by real-time RT-PCR. Antileukemic induction therapy consisted of idarubicin and cytarabine, with or without the addition of fludarabine. Among patients who received antileukemic therapy and for whom survival information was available, 17 of 34 (50%) achieved a complete remission (CR) with initial induction therapy; 10 (29%) patients required additional induction therapy to achieve CR, while 7 (21%) patients failed to achieve remission. Of 27 patients who achieved CR, 11 (41%) experienced subsequent leukemic relapse. Thus, our patient cohort experienced clinical outcomes representative of the modern experience with adult AML.The results of ABCG2 mRNA quantification can be divided into 3 categories: low (lower than normal PB and BM), intermediate (greater
than PB and BM but lower or equal to clone 3.3), and high (greater than
clone 3.3). Of the patients, 18% had low ABCG2 mRNA levels,
ranging from 0.01% to 2.0% of the levels in clone 3.3 (Figure
6); 75% had intermediate ABCG2 mRNA
levels (2.1 to 100% of clone 3.3). Three patients (7%) had high ABCG2
mRNA levels, ranging from 140% to 2600% of that seen in clone
3.3. Two of these had ABCG2 mRNA levels exceeding the level in
clone 6.2. These high-expressing samples showed no PCR amplification
when reverse transcriptase was not included in the reaction, indicating
that the high ABCG2 levels were not the result of contamination by DNA.
Patient characteristics and results of real-time RT-PCR analysis of
patient samples for ABCG2 mRNA expression are summarized in Table 1.
There were no obvious correlations
between ABCG2 expression and clinical parameters or outcomes. While the
percentage of blast cells in patient samples ranged from 31% to 98%,
mathematical correction of the ABCG2 mRNA levels for the proportion on
normal cells did not significantly change the category groupings.
Altogether, these results showed that most cases of AML had ABCG2 mRNA
levels that were significantly greater than those in normal bone marrow or peripheral blood, but that rarely approached the levels associated with our drug-resistant 6.2 clone.
ABCG2 protein expression in AML samples by flow cytometry In order to determine the cellular distribution of ABCG2 expression within AML samples, we analyzed several available AML specimens by flow cytometry after labeling with the 5D3 anti-ABCG2 monoclonal antibody. In many cases, the viability was poor after thawing of these frozen samples, as indicated by PI staining. In 3 cases with a sufficient number of viable cells, very small subpopulations of ABCG2-positive cells were identified, which represented 2.6%, 0.4%, and 1.8% of total mononuclear cells for patients 8, 16, and 38, respectively (Figure 7). The ratio of 5D3 to isotype mean fluorescent intensity was 3.7:1, 1.1:1, and 4.1:1 for patients 8, 16, and 38, respectively. These results suggest that the ABCG2 mRNA expression levels detected by real-time RT-PCR were due to minor subpopulations of ABCG2-expressing cells, rather than a homogenous distribution of leukemic cells with very low levels of ABCG2 expression.
While ABCG2 expression in AML recently has been reported in several studies, our study addresses several important gaps in knowledge regarding this area. ABCG2 mRNA expression has been documented with RT-PCR in a number of cases,6 but it was not clear whether these levels of expression were significant with regard to drug resistance or if expression could have been due to contaminating normal hematopoietic cells. Another study measured ABCG2 protein expression in blast cell samples but did not indicate what proportion of AML cells was expressing the ABCG2 transporter.21 Another question is whether the ABCG2 protein can confer resistance to idarubicin, a commonly used induction-phase drug for treating AML. Based on our analysis, a majority of AML patient samples showed an intermediate level of ABCG2 mRNA expression. This level was well below that shown to correlate with drug resistance and detectable surface protein but was significantly higher than the background levels in normal peripheral blood and bone marrow. Therefore, these intermediate levels cannot be explained by the presence of normal blood or bone marrow cells within the leukemic patient samples. There are 2 main scenarios that could explain these findings. First, ABCG2 could have been expressed homogenously among leukemic cells at low levels. Based on the measured mRNA levels in these intermediate samples, a homogenous pattern of ABCG2 expression would predict cellular expression levels below that seen in clone 3.3 and would therefore be insufficient to confer drug resistance or mitoxantrone efflux. Under this scenario, we would not expect significant drug resistance in these intermediate AML cases. Another possible explanation for the frequent intermediate expression levels is that ABCG2 expression could be heterogenous and limited to a subpopulation of leukemic cells. Our antibody staining results show that this latter scenario was true in all cases we examined by flow cytometry. In these cases, a small subpopulation of ABCG2-expressing cells were the main source for the ABCG2 mRNA signal detected in the blast cell samples. This suggests that the expression per cell in this small subpopulation was relatively high and could be above the threshold required for drug resistance. This could be clinically significant with regard to drug resistance if these rare cells were clonogenic leukemic stem cells. There are several other lines of evidence to suggest that leukemic stem cells could express high levels of ABCG2. Several studies have shown both normal and leukemic cells within the SP fraction of AML bone marrow.14,15 Furthermore, the side population is often expanded in leukemic marrow.14 Because ABCG2 expression is a critical molecular determinant of the SP phenotype in normal stem cells,8 it is possible that leukemic stem cells also share this molecular phenotype. This idea is consistent with the recent report showing that ABCG2 protein expression was positively correlated with a primitive immunophenotype within AML blasts samples.21 Such a subpopulation might represent a reservoir of primitive leukemic stem cells with intrinsic drug efflux capacity and could potentially contribute to subsequent relapse in certain cases. This would be consistent with the recent finding of increased ABCG2 mRNA levels at relapse compared to initial diagnosis.22 Recently, idarubicin and mitoxantrone have become increasingly used in the remission induction therapy for AML. To our knowledge, this is the first report to demonstrate the lack of idarubicin transport or drug resistance by ABCG2. In contrast, mitoxantrone is readily effluxed by ABCG2-expressing cells, which also displayed significant mitoxantrone drug resistance. Taken together, these results suggest that idarubicin could have a clinical advantage over mitoxantrone in cases where AML stem cells express significant levels of ABCG2.
We thank Richard Ashmun for skillful assistance with flow cytometry. For assistance with procurement of normal hematopoietic cells from volunteer donors, we thank Rupert Handgretinger, Paul Gordon, and Patrick Kelly. We are grateful to the patients and medical and nursing staff of Baptist Memorial Hospital in Memphis, TN, for assistance with the procurement of cord blood samples.
Submitted January 29, 2002; accepted July 17, 2002.
Prepublished online as Blood First Edition Paper, July 25, 2002; DOI 10.1182/blood-2002-01-0271.
Supported in part by grant R01-HL67366-01 (B.P.S.) and P01-CA55164 (M.A.) from the National Institutes of Health; the A.S.S.I.S.I. foundation of Memphis grant 94-00 (B.P.S.); the Cancer Center Support Grant P30-CA21765; and the American-Lebanese-Syrian Associated Charities (A.L.S.A.C.).
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.
Reprints: Brian P. Sorrentino, Division of Experimental Hematology, St Jude Children's Research Hospital, 332 North Lauderdale Ave, Memphis, TN 38105; e-mail: brian.sorrentino{at}stjude.org.
1.
Ueda K, Cardarelli C, Gottesman MM, Pastan I.
Expression of a full-length cDNA for the human "MDR1" gene confers resistance to colchicine, doxorubicin, and vinblastine.
Proc Natl Acad Sci U S A.
1987;84:3004-3008 2. Del Poeta G, Venditti A, Aronica G, et al. P-glycoprotein expression in de novo acute myeloid leukemia. Leuk Lymphoma. 1997;27:257-274[Medline] [Order article via Infotrieve]. 3. Zhou DC, Zittoun R, Marie JP. Expression of multidrug resistance-associated protein (MRP) and multidrug resistance (MDR1) genes in acute myeloid leukemia. Leukemia. 1995;9:1661-1666[Medline] [Order article via Infotrieve].
4.
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.
1999;94:1086-1099
5.
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.
1998;95:15665-15670
6.
Ross DD, Karp JE, Chen TT, Doyle LA.
Expression of breast cancer resistance protein in blast cells from patients with acute leukemia.
Blood.
2000;96:365-368 7. Sargent JM, Williamson CJ, Maliepaard M, Elgie AW, Scheper RJ, Taylor CG. Breast cancer resistance protein expression and resistance to daunorubicin in blast cells from patients with acute myeloid leukaemia. Br J Haematol. 2001;115:257-262[CrossRef][Medline] [Order article via Infotrieve]. 8. 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. 2001;7:1028-1034[CrossRef][Medline] [Order article via Infotrieve].
9.
Scharenberg CW, Harkey MA, Torok-Storb B.
The ABCG2 transporter is an efficient Hoechst 33342 efflux pump and is preferentially expressed by immature human hematopoietic progenitors.
Blood.
2002;99:507-512 10. 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. 2000;113:2011-2021[Abstract].
11.
Honjo Y, Hrycyna CA, Yan QW, et al.
Acquired mutations in the MXR/BCRP/ABCP gene alter substrate specificity in MXR/BCRP/ABCP-overexpressing cells.
Cancer Res.
2001;61:6635-6639
12.
Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC.
Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo.
J Exp Med.
1996;183:1797-1806 13. Goodell MA, Rosenzweig M, Kim H, et al. Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat Med. 1997;3:1337-1345[CrossRef][Medline] [Order article via Infotrieve].
14.
Wulf GG, Wang RY, Kuehnle I, et al.
A leukemic stem cell with intrinsic drug efflux capacity in acute myeloid leukemia.
Blood.
2001;98:1166-1173
15.
Feuring-Buske M, Hogge DE.
Hoechst 33342 efflux identifies a subpopulation of cytogenetically normal CD34(+)CD38(-) progenitor cells from patients with acute myeloid leukemia.
Blood.
2001;97:3882-3889 16. Wang C, Curtis JE, Minden MD, McCulloch EA. Expression of a retinoic acid receptor gene in myeloid leukemia cells. Leukemia. 1989;3:264-269[Medline] [Order article via Infotrieve]. 17. Davis L, Kuehl M, Battey J. Basic Methods in Molecular Biology. 2nd ed. Norwalk, CT: Appleton and Lange; 1994.
18.
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.
2001;61:3458-3464 19. Larochelle A, Vormoor J, Hanenberg H, et al. Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy. Nat Med. 1996;2:1329-1337[CrossRef][Medline] [Order article via Infotrieve]. 20. de Wynter EA, Buck D, Hart C, et al. CD34+ AC133+ cells isolated from cord blood are highly enriched in long-term culture-initiating cells, NOD/SCID-repopulating cells and dendritic cell progenitors. Stem Cells. 1998;16:387-396[Medline] [Order article via Infotrieve].
21.
Van Der Kolk DM, Vellenga E, Scheffer GL, et al.
Expression and activity of breast cancer resistance protein (BCRP) in de novo and relapsed acute myeloid leukemia.
Blood.
2002;99:3763-3770 22. Van Den Heuvel-Eibrink MM, Wiemer EA, Prins A, et al. Increased expression of the breast cancer resistance protein (BCRP) in relapsed or refractory acute myeloid leukemia (AML). Leukemia. 2002;16:833-839[CrossRef][Medline] [Order article via Infotrieve].
© 2002 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  C. Ozvegy-Laczka, R. Laczko, C. Hegedus, T. Litman, G. Varady, K. Goda, T. Hegedus, N. V. Dokholyan, B. P. Sorrentino, A. Varadi, et al. Interaction with the 5D3 Monoclonal Antibody Is Regulated by Intramolecular Rearrangements but Not by Covalent Dimer Formation of the Human ABCG2 Multidrug Transporter J. Biol. Chem., September 19, 2008; 283(38): 26059 - 26070. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. L. Guzman, X. Li, C. A. Corbett, R. M. Rossi, T. Bushnell, J. L. Liesveld, J. Hebert, F. Young, and C. T. Jordan Rapid and selective death of leukemia stem and progenitor cells induced by the compound 4-benzyl, 2-methyl, 1,2,4-thiadiazolidine, 3,5 dione (TDZD-8) Blood, December 15, 2007; 110(13): 4436 - 4444. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Wang, L.-P. Guo, L.-Z. Chen, Y.-X. Zeng, and S. H. Lu Identification of Cancer Stem Cell-Like Side Population Cells in Human Nasopharyngeal Carcinoma Cell Line Cancer Res., April 15, 2007; 67(8): 3716 - 3724. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. E. Jordanides, H. G. Jorgensen, T. L. Holyoake, and J. C. Mountford Functional ABCG2 is overexpressed on primary CML CD34+ cells and is inhibited by imatinib mesylate Blood, August 15, 2006; 108(4): 1370 - 1373. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Benderra, A. M. Faussat, L. Sayada, J.-Y. Perrot, R. Tang, D. Chaoui, H. Morjani, C. Marzac, J.-P. Marie, and O. Legrand MRP3, BCRP, and P-Glycoprotein Activities are Prognostic Factors in Adult Acute Myeloid Leukemia Clin. Cancer Res., November 1, 2005; 11(21): 7764 - 7772. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. H. Kaufmann, J. E. Karp, L. Letendre, T. J. Kottke, S. Safgren, J. Greer, I. Gojo, P. Atherton, P. A. Svingen, D. A. Loegering, et al. Phase I and Pharmacologic Study of Infusional Topotecan and Carboplatin in Relapsed and Refractory Acute Leukemia Clin. Cancer Res., September 15, 2005; 11(18): 6641 - 6649. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. T. Budak, O. S. Alpdogan, M. Zhou, R. M. Lavker, M.A. M. Akinci, and J. M. Wolosin Ocular surface epithelia contain ABCG2-dependent side population cells exhibiting features associated with stem cells J. Cell Sci., April 15, 2005; 118(8): 1715 - 1724. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Sorrentino The role of Abcg2 expression in hematopoietic stem cells AACR Meeting Abstracts, April 1, 2005; 2005(1): 1476 - 1476. [Abstract]
|
|
|
|
|
|
M. H.G.P. Raaijmakers, E. P.L.M. de Grouw, L. H.H. Heuver, B. A. van der Reijden, J. H. Jansen, R. J. Scheper, G. L. Scheffer, T. J.M. de Witte, and R. A.P. Raymakers Breast Cancer Resistance Protein in Drug Resistance of Primitive CD34+38- Cells in Acute Myeloid Leukemia Clin. Cancer Res., March 15, 2005; 11(6): 2436 - 2444. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Zhou, Y. Zong, P. A. Ney, G. Nair, C. F. Stewart, and B. P. Sorrentino Increased expression of the Abcg2 transporter during erythroid maturation plays a role in decreasing cellular protoporphyrin IX levels Blood, March 15, 2005; 105(6): 2571 - 2576. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Ozvegy-Laczka, G. Varady, G. Koblos, O. Ujhelly, J. Cervenak, J. D. Schuetz, B. P. Sorrentino, G.-J. Koomen, A. Varadi, K. Nemet, et al. Function-dependent Conformational Changes of the ABCG2 Multidrug Transporter Modify Its Interaction with a Monoclonal Antibody on the Cell Surface J. Biol. Chem., February 11, 2005; 280(6): 4219 - 4227. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Benderra, A.-M. Faussat, L. Sayada, J.-Y. Perrot, D. Chaoui, J.-P. Marie, and O. Legrand Breast Cancer Resistance Protein and P-Glycoprotein in 149 Adult Acute Myeloid Leukemias Clin. Cancer Res., December 1, 2004; 10(23): 7896 - 7902. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Suarez, M.-B. Vidriales, J. Garcia-Larana, G. Sanz, M.-J. Moreno, A. Lopez, S. Barrena, R. Martinez, M. Tormo, L. Palomera, et al. CD34+ Cells from Acute Myeloid Leukemia, Myelodysplastic Syndromes, and Normal Bone Marrow Display Different Apoptosis and Drug Resistance-Associated Phenotypes Clin. Cancer Res., November 15, 2004; 10(22): 7599 - 7606. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Krishnamurthy, D. D. Ross, T. Nakanishi, K. Bailey-Dell, S. Zhou, K. E. Mercer, B. Sarkadi, B. P. Sorrentino, and J. D. Schuetz The Stem Cell Marker Bcrp/ABCG2 Enhances Hypoxic Cell Survival through Interactions with Heme J. Biol. Chem., June 4, 2004; 279(23): 24218 - 24225. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
Ct was subtracted from the 
[ABCG2 mRNA level in normal bone
marrow × (1
indicates
OCI-AML3; 
, clone 3.3;
, clone 6.2. Clones
were incubated with continuous drug exposure at the indicated
concentrations for 96 hours, at which time cell viability was assessed
using a modified MTT reagent. Results are expressed as percentage of
cells viable relative to the initial number of cells. Error bars
represent the SD from 6 replicates of each dose.
and AC133
