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Leukemia (1997) 11, 48–53
 1997 Stockton Press All rights reserved 0887-6924/97 $12.00
A novel energy dependent mechanism reducing daunorubicin accumulation in acute
myeloid leukemia
DW Hedley1, S Xiu-Yan Xie1, MD Minden1, CH Choi1,2, H Chen1,3 and V Ling1,4
1
Ontario Cancer Institute/Princess Margaret Hospital, Department of Medical Biophysics, University of Toronto, 610 University Avenue,
Toronto, Ontario M5G 2M9, Canada
Using cyclosporin A (CsA) to inhibit P-glycoprotein (P-gp) function we showed previously that there was a discordance
between the ability of acute myeloid leukemic (AML) blast cells
to accumulate daunorubicin and P-gp antigen expression (Xie
et al, Leukemia 1995; 9: 1882–1887). This discordance suggests
that a CsA-sensitive drug efflux mechanism distinct from P-gp
is expressed in many clinical samples. In the present study
using the ATP depleting agents cyanide, azide, or dinitrophenol
to inhibit energy dependent transport processes, we observed
even larger increases in daunorubicin accumulation than were
seen with CsA. Similar patterns were seen in a wide range of Pgp negative human cancer cell lines. Also the observed cyanide
effect did not correlate with the expression of mRNA for multidrug resistance-associated protein (MRP), the only other member of the ABC family of membrane transporters that is known
to be capable of effluxing daunorubicin. Thse results suggest
that daunorubicin accumulation in many cases of AML is
modulated by one or more novel energy-dependent processes
that are distinct from P-gp or MRP. We speculate that this novel
drug transport mechanism(s) may influence the response of
AML patients to daunorubicin and other therapeutic agents.
Keywords: leukemia; drug resistance; P-glycoprotein; MRP; ABC
transporter
Introduction
P-glycoprotein is a member of a large class of membrane
transporters coded for by the ATP-binding cassette (ABC) gene
superfamily.1,2 The physiological function of most of these
ABC transporter gene products is unknown, but an additional
member, multidrug resistance associated protein (MRP), has
recently been shown to produce resistance to a range of natural products such as anthracyclines and vinca alkaloids by an
active transport mechanism similar to that of P-gp.3,4 Although
expression of MRP has been observed in leukemic blasts its
clinical significance is still unknown.5 When we compared
the amount of P-gp antigen expressed on the cell membrane
to the modulating effects of the P-gp inhibitor cyclosporin A
(CsA) on daunorubicin accumulation, we found little correlation between the two assays in a series of AML patients.6
Compared to the results obtained with P-gp expressing cell
lines, the AML samples showed generally greater transport
function in relation to Pgp antigen expression. This suggests
that the modulating effect of CsA on daunorubucin accumulation, commonly regarded as a P-gp functional assay, can
detect drug efflux mechanisms that are antigenically distinct
from classical Pg-p. Because CsA might be a poor inhibitor of
some of these putative non-P-gp transporters, the functional
assay was modified by replacing CsA with agents that inhibit
Correspondence: DW Hedley
Present addresses: 2Department of Pharmacology, Chosun University,
Kwangju City, Korea; 3Allelix Corporation, Mississauga, Ontario, Canada; 4BC Cancer Research Centre, 601 West 10th Avenue, Vancouver,
BC V5Z 1L3, Canada
Received 30 May 1996; accepted 27 September 1996
oxidative phosphorylation, such as cyanide and azide, thereby
depleting cellular ATP and inhibiting all energy-dependent
processes. This strategy was used originally to demonstrate the
ATP dependence of P-gp,7 and more recently has been shown
to inhibit drug efflux via MRP.8,9 We now report that it also
appears to show the presence of high levels of non-P-gp
mediated daunorubicin efflux in cell lines and blast cells
obtained from patients with acute myeloid leukemia.
Materials and methods
Cell lines
The following human cell lines were used to study energydependent daunorubicin efflux: CCRF-CEM T lymphoblasts,
the vinblastine-resistant lines VLB 0.1 and VLB 1.0,8 and the
AML lines OCI/AML 1–5, established in this Institute. In
addition, we investigated the human carcinoma lines: MGHU1 (bladder), MCF7 (breast), SKOV3 (ovary), and HCT8
(colon). Parental HeLaS3 cervical carcinoma cells, and the
multidrug resistance-associated protein (MRP) expressing variant HeLaJ2,4 were kindly supplied by Dr Raymond Baker,
Roswell Park Cancer Institute, Buffalo, NY, USA. With the
exception of the CCRF-CEM VLB 0.1 and 1.0 cells, none of
these lines showed increased expression of P-glycoprotein as
assessed using Western blotting with monoclonal antibody
C219,10 or surface immunolabelling with the MRK-16 antibody.11
Clinical samples
Peripheral blood samples containing at least 50% leukemic
blasts were obtained from 49 patients with acute myeloid leukemia, either at initial presentation (27 patients) or with recurrent disease following prior chemotherapy (22 patients).
Induction chemotherapy consisted of a standard protocol of
high dose cytosine arabinoside and daunorubicin. Blasts were
separated by density gradient centrifugation, washed, counted
and resuspended in PBS at 1 × 106/ml.
Flow cytometric measurement of P-gp
Cell surface expression of Pgp was detected using the MRK16 antibody (gift of Dr T Tsuruo, University of Tokyo), which
recognizes an external epitope of the human MDR-1 gene
product.11 Because epitope masking can impair labelling of
AML blasts with this antibody,6,12 cells were incubated with
0.2 U neuraminidase (type V, from Clostridium perfringens;
Sigma Chemicals, St Louis, MO, USA) per 2 × 106 cells in 1
ml at 37°C for 30 min, and washed in PBS prior to antibody
labelling. Cells were resuspended at 1 × 107/ml in PBS, and
Energy-dependence of daunorubicin uptake in AML
DW Hedley et al
incubated for 30 min with a 1:10 dilution of goat gammaglobulin on ice, to block non-specific binding sites. They were
then incubated with 20 mg/ml MRK-16 antibody for a further
30 min on ice. An MRK-16 dilution curve using CCRF-CEM
VLB 0.1 cells showed that this was a saturating concentration
of antibody. After washing twice with cold PBS, the cells were
incubated with a 1:25 dilution of phycoerythrin (PE)-conjugated F(ab′)2 goat anti-mouse immunoglobulin (Dakopatts,
Denmark) on ice for 30 min, and washed twice.6
Fluorescence calibration beads were used to obtain an estimate of the number of antigenic sites per cell. These calibration beads (SpheroTech, Libertyville, IL, USA), consist of
a mixture of particles at six different fluorescent intensities,
calibrated to give the mean equivalent soluble fluorochrome
(MESF). Thus the mean number of phycoerythrin molecules
bound to the cell surface can be estimated.
Flow cytometric measurement of daunorubicin
accumulation
Cells were incubated in 3 mg/ml daunorubicin, which is naturally fluorescent, at a concentration of 1 × 106 cells/ml, ±3
mg/ml CsA at 37°C for 30 min. Increased efflux of daunorubicin by Pgp causes a decrease in daunorubicin accumulation
that can be reversed by the addition of CsA. The mean fluorescence from each daunorubucin and daunorubucin + CsA
sample was recorded, and the Pgp functional activity
expressed as the percentage increase in daunorubicin fluorescence after CsA treatment, ie:
(mean fluorescence + CsA) − (mean fluorescence − CsA)
× 100.
(mean fluorescence − CsA)
In addition to CsA, which is a classical P-gp inhibitor, cellular
ATP was depleted by the addition of 2 mm KCN, 2 mm NaN3,
or 1 mm dinitrophenol, in order to inhibit all energy-dependent drug transport mechanisms. Thus a % functional activity
(KCN etc) was calculated similar to the % functional activity
(CsA).
Flow cytometry was done using an Epics Elite cell sorter
(Coulter Electronics, Hialeah, FL, USA) equipped with an aircooled argon laser emitting at 488 nm and operating at 15
mW. Forward and side scatter signals were collected using
linear amplification to define the blast population, from which
the fluorescence data were taken. Phycoerythrin fluorescence
was measured with a 575 nm bandpass filter using logarithmic
amplification, and daunorubicin fluorescence with the same
filter using linear amplification. A minimum of 20 000 events
were collected for each sample.
actin cDNA, the original probe was removed by boiling the
blots in 0.5% SDS for 5 min. Expression of MRP in human
cell lines was measured using Northern blotting. Results were
compared to the level of b actin expression, and for comparison between the cell lines they were normalized with the ratio
MRP:b action for HeLaS3 cells set as 1.
Results
Measurement of P-gp function and antigen expression
in acute leukemia samples
Using the methods described above, we have previously
found that in the blast cells of the majority of AML patients,
P-gp antigen expression is detectable, and daunorubicin
accumulation is increased in the presence of CsA, an inhibitor
of P-gp function. Unexpectedly, there appeared to be little
correlation between the amount of P-gp detected and the
functional assay.6 In the present study we used fluorescent
calibration beads to quantitate the number of phycoerythrin
molecules bound per cell, thereby obtaining an estimate to
the actual number of P-gp antigenic sites. Although the stoichiometry of the immunolabelling technique is unlikely to be
strictly 1:1, the error introduced is small in relation to the
range of values seen between individual samples, and the use
of standardized measurement units facilitates comparison
between the levels of P-gp found in different cell populations.
As seen in Figure 1, there is a clear correlation between transport function and antigen expression in a series of CCRF-CEM
human T lymphoblast lines, which are well characterized with
regard to their P-gp expression. When the data for the cell
lines and AML samples from patients were plotted using the
same axis scales, it was observed that compared to the cell
lines, the clinical samples showed relatively low levels of
Measurement of Pgp and MRP mRNA
Cells grown at exponential stage were harvested and subjected to total RNA isolation using the guanidinium isothiocyanate method according to Chomczynski and Sacchi.13 Total
RNA 15 mg was then size fractionated on a denaturing formaldehyde agarose (1%) gel, transferred to Hybond N Membrane
(Amersham, Arlington Heights, IL, USA), and probed with a
(32P) labelled MRP cDNA obtained from Dr Susan Cole,
Queen’s University, Kingston, Ontario, Canada. (Fragment
10.1, comprising 1 kb from the 3′ end of MRP, including the
second nucleotide binding domain, 1–5 × 105 c.p.m./ml, 105
c.p.m./mg, random priming method.) For reprobing with b-
Figure 1
Correlation between the percentage increase in daunorubicin accumulation with CsA and P-gp antigen expression determined
by quantitative MRK-16 labelling. CCRF-CEM lines (I): CEM wildtype, CEM VLB 0.1 and CEM VLB 1.0; blasts from AML patients (o).
Data replotted from Ref. 6.
49
Energy-dependence of daunorubicin uptake in AML
DW Hedley et al
50
MRK-16 labelling but some of the samples showed disproportionately high increases in daunorubicin accumulation
with CsA, relative to the number of P-gp antigenic sites per
cell (Figure 1). This suggests that in a significant proportion of
AML patients, CsA-inhibitable daunorubicin efflux can occur
by mechanisms that are antigenically distinct from P-gp.
It is now established that energy-dependent efflux of daunorubicin can result from overexpression of MRP, another member of the ABC family of membrane transporters.8,14 Furthermore, MRP expression has recently been reported in some
AML patients.5,15 MRP is less sensitive than P-gp to CsA inhibition.8 We were therefore interested in determining if the
concentration of CsA used in our experiments would be effective against an MRP-mediated reduction in daunorubicin
accumulation. We used the well-characterized HeLaJ2 cell
line containing an amplified MRP gene4 to investigate this
question. We observed no measurable increase in daunorubicin accumulation when these cells were treated with 3 mg/ml
CsA (data not shown). Therefore, the CsA-stimulated accumulation of daunorubicin observed in the AML samples is not
likely due to expression of MRP.
Effects of metabolic inhibitors on the accumulation of
daunorubicin by cell lines
In addition to agents such as CsA or verapamil, that probably
act by competitive inhibition of substrate binding sites, drug
efflux via P-gp can be reversed using ATP-depleting agents
such as cyanide or azide. This strategy was originally used by
Ling and colleagues7 to show the energy dependence of P-gp.
More recently, depletion of cellular ATP has also been shown
to inhibit MRP,8,9 and is likely to inhibit all other energydependent processes influencing daunorubicin accumulation.
In other words, substitution of ATP-depleting agents for CsA
in the daunorubicin accumulation assay will give a summation of all energy-dependent drug transport processes,
regardless of whether these are sensitive to classical P-gp
inhibitors such as CsA or verapamil.
Initially, the five human AML cell lines OCI/AML 1–5 were
investigated, since these have been extensively characterized
in our Institution, and express very low levels of P-gp. This
was confirmed in the present series of experiments by demonstrating a lack of MRK-16 staining and low levels of CsAdependent increases in daunorubicin accumulation in all five
lines. Cells were treated with 2 mm KCN, 2 mm sodium azide,
or 1 mm dinitrophenol (DNP) in glucose-free medium, in an
assay that was otherwise identical to the P-gp functional assay
described above. Similar results were obtained for all five
AML lines, with a mean increase in daunorubicin accumulation of 122% ± 25 s.d. using KCN, compared to 6% ± 4 s.d.
when CsA was used as the modulating agent. Similar results
were obtained for all three metabolic inhibitors, and for this
reason most subsequent experiments were done using 2 mm
KCN only.
In order to establish that the increases in daunorubicin
accumulation seen with KCN treatment were due to the inhibition of an ATP-dependent efflux mechanism, 5 mm glucose
was added at the end of the 30 min incubation period. The
cells were maintained in daunorubicin ± KCN throughout the
experiment. In the presence of continued exposure to KCN,
glucose allows ATP generation to take place by glycolysis.7
As shown in Figure 2, addition of glucose to cells pre-loaded
with daunorubicin in the presence of KCN resulted in the
rapid loss of drug, so that by 20 min daunorubicin fluor-
Figure 2
Reversal of KCN effect by addition of glucose. AML1
cells were preloaded with daunorubicin in the presence (k) or the
absence (K) of 2 mm KCN. At time = 0, glucose 5 mm was added in
order to allow ATP generation by glycolysis. Note that daunorubicin
fluorescence rapidly falls to that of cells loaded with drug in the
absence of KCN.
escence values were similar to those of controls that were preloaded with daunorubicin in the absence of KCN. This reversal effect of glucose is consistent with our hypothesis that KCN
treatment inhibits an ATP-dependent daunorubicin efflux
mechanism.
In addition to active extrusion via P-gp, cellular accumulation of weak bases such as daunorubicin may be influenced
by the pH gradient across the surface membrane.16,17 A relative reduction in intracellular pH may cause decreased drug
accumulation, because the equilibrium is shifted to the protonated form which diffuses much more slowly through lipid
bilayers. To test the possibility that KCN was causing
increased daunorubicin accumulation by producing cellular
acidosis, thereby altering drug partitioning across the surface
membrane, cells were loaded with the pH-sensitive dye
SNARF-1, and relative intracellular pH values measured using
a standard ratio metric flow cytometry assay.18 Within the sensitivity of the assay (approximately 0.2 pH units) there was
no detectable change in intracellular pH during the 30 min
exposure to KCN, suggesting that acidification of cytoplasm is
unlikely to account for the increased daunorubicin uptake.
KCN-sensitive daunorubicin accumulation and MRP
expression in P-gp-negative human cell lines
The increased daunorubicin accumulation seen in the
OCI/AML cell lines following KCN treatment suggests that
energy-dependent mechanisms other than classical P-gp are
expressed in these cells, and inhibit drug uptake. To date the
Energy-dependence of daunorubicin uptake in AML
DW Hedley et al
only other ABC transporter known to be expressed in human
cells and capable of transporting daunorubicin is MRP. We
therefore compared the expression of MRP to the effect of
KCN on daunorubicin accumulation. In addition to the
OCI/AML lines, we examined parental CCRF-CEM T lymphoblasts, and a series of human carcinoma lines including parental HeLa cells and the MRP overexpressing line HeLaJ2.
None of these cell lines overexpresses P-gp. Expression of
MRP was determined by comparing levels of mRNA for MRP
to those for b actin, using Northern blotting, these results then
being normalized to MRP expression by parental HeLaS3
cells, which was set an an arbitrary value of 1. As shown in
Figure 3, there was a wide range of values between the 12
cell lines. As expected, the highest level of MRP expression
was found in HeLaJ2 cells, but significant levels were also
seen in the OCI/AML lines AML1 and AML4, and in the HCT8
colon cancer line. In all cases, daunorubicin uptake was
increased by co-incubation with 2 mm KCN, but there was no
correlation with the level of MRP expression. The results for
the OCI/AML lines were particularly striking. As illustrated in
Figure 3, despite expressing very low levels of MRP mRNA,
the lines AML2, AML3 and AML5 showed similar increases in
daunorubicin uptake with KCN treatment to those seen in the
MRP expressing lines AML1 and AML4.
Comparative effects of CsA and KCN on daunorubicin
uptake by AML blasts
A comparison was made between the effects of CsA and KCN
on daunorubicin accumulation by leukemic blasts obtained
from patients. In some clinical samples the effects of KCN
Figure 3
Correlation between the MRP mRNA level and percentage increase in daunorubicin accumulation after KCN treatment in a
series of low P-gp expressing human leukemic and epithelial tumor
cell lines.
were comparable to those seen with CsA, suggesting that Pgp
overexpression or some other non-P-gp but CsA-sensitive
mechanism was a major determinant of the reduced daunorubicin uptake. In others, the increase was much greater with
KCN suggesting the presence of both CsA-responsive and nonresponsive energy-dependent daunorubicin transport mechanisms (Figure 4). These results are summarized in Figure 5.
In the majority of cases, accumulation of daunorubicin was
increased in the presence of 2 mm KCN. The magnitude of
this effect varied widely between patients, ranging from no
significant effect to .200% increases in daunorubicin
accumulation. The trend was for larger increases in drug
uptake with KCN treatment in patients with recurrent disease
following previous chemotherapy using daunorubicin-containing regimens compared to patients at initial diagnosis
(mean increase 123% ± 51 s.d., compared to 95% ± 57 s.d.;
P . 0.05 using Student’s t-test).
Discussion
Using neuraminidase pretreatment and a sensitive immunofluorescence technique, P-gp could be detected in almost all
cases of AML. However, the levels of expression were 30–100
times lower than those seen in P-gp overexpressing CCRFCEM cell lines. Considerable interpatient heterogeneity was
seen in the levels of both P-gp antigen expression, and in
cyclosporin-inhibitable daunorubicin efflux. Although the latter is widely taken to be a measure of P-gp functional activity,
there was no significant correlation with P-gp antigen
expression by AML leukemic blasts.6 This finding is consistent
with some other recent reports for AML,19,20 and in contrast
to chronic lymphocytic leukemia, where the daunorubicin
accumulation assay has been shown to correlate closely with
the levels of P-gp antigen and mRNA expression.12,21
Replotting the AML patient results on the same axis scale
as that for the P-gp expressing cell lines shows that in many
cases the patient samples have much higher activity in the
functional assay than would be predicted from the amount of
P-gp antigen on the cell surface (Figure 1). One explanation
of this finding might be overexpression of MRP, the only other
ABC transporter that is known to be expressed in AML and to
be capable of effluxing daunorubicin. However, CsA is a poor
inhibitor of MRP. Using a concentration of 3 mm CsA we
found no significant increases in uptake of daunorubicin by
the MRP amplified line HeLaJ2, or the leukemic lines
OCI/AML1 and 4, both of which showed considerable MRP
Figure 4
Overlay histograms showing uptake of daunorubicin by
leukemic blasts from an AML patient, comparing the effects of CsA
and KCN to control values.
51
Energy-dependence of daunorubicin uptake in AML
DW Hedley et al
52
Figure 5
Comparison between the modulating effects of CsA and KCN on daunorubicin accumulation by AML blasts obtained from: (a)
patients at initial diagnosis, and (b) patients with recurrent disease following previous chemotherapy.
expression by Northern blotting. A more likely explanation is
that a novel CsA-sensitive mechanism is reducing daunorubicin accumulation in many AML samples.
A large number of ABC transporters have now been identified in human cells on the basis of sequence homologies in
the highly conserved ATP binding region.1 The function of
most of these is unknown. Because of the possibility that an
additional ABC transporter might be active in some AML
samples, the functional assay was modified using ATP-depleting agents in order to inhibit all energy-dependent processes.
This strategy was originally used to demonstrate the ATP
dependence of P-gp,7 and has recently been shown to inhibit
MRP.8,22 It is likely to provide a broader spectrum of activity
than agents such as CsA that inhibit substrate binding sites.
Generalized ATP depletion resulted in large increases in
daunorubicin accumulation by a wide range of human cell
lines, including some that express little or no P-gp or MRP.
Addition of glucose, which allows ATP generation by glycolysis, reversed this effect. Alterations in intracellular pH have
been shown to affect cellular accumulation and subcellular
distribution of weakly basic drugs such as daunorubicin.16
However, no significant changes in intracellular pH were
detected during 30 min exposure to 2 mm KCN. The most
likely explanation for these findings is that ATP depletion
inhibits an energy-dependent process that reduces daunorubicin accumulation.
Comparison of the modulating effects of CsA and KCN in
the AML samples shows considerable interpatient heterogeneity (Figure 5). In some cases neither agent increased drug
accumulation, indicating that no energy-dependent processes
were active. In others the effects of CsA and KCN were
roughly comparable, suggesting the expression of P-gp or a
closely related transporter. In striking contrast, however, some
patient samples showed very little modulation of daunorubicin uptake by CsA, but large increases with KCN. We propose
that when KCN is used as the modulating agent, the daunorubicin accumulation assay sums the activities of all energydependent processes that reduce drug uptake, whether these
be ABC transporters, or other mechanisms. If this interpretation is correct, then some AML patient samples show daunorubicin efflux rates that are similar to those seen in CCRF-CEM
VLB 1.0 cells expressing .100 000 P-gp antigenic sites per
cell (see Figure 1). We do not believe that the energy-dependent drug transport phenomena observed in the leukemia
patients can be explained simply on the basis of variable
expression of P-gp and MRP, since large increases were seen
in daunorubicin accumulation following KCN treatment were
also seen in AML lines that express very low levels of P-gp
and MRP. More likely, this study suggests the presence of a
novel mechanism(s) in some patient blast cells.
Identification of new drug resistance mechanisms usually
involves the use of cell lines selected by in vitro drug
exposure. Stepwise increases in drug concentrations over time
can result in very high levels of resistance to agents such as
anthracyclines or vinca alkaloids, frequently associated with
increased P-gp or MRP expression. However, drug selection
is clearly not essential for this since elevated P-gp or MRP
levels are also seen in some untreated cancer patients, possibly because the cancer is derived from a normal tissue that
naturally expresses the resistance mechanism, or as a genetic
alteration occurring as part of the neoplatic process. It is now
recognized that the ABC transporter family is extremely
diverse in nature,1 and that several members are capable of
conferring drug resistance in a wide range of species. The
human genome may contain many ABC transporters which
have not yet been identified and it would not therefore be
surprising if some of these as yet uncharacterized ABC transporters were constitutively expressed in AML blasts, and capable of transporting daunorubicin.
The constitutive expression of high levels of non-P-gp drug
transporters might be one explanation for the comparatively
weak association between increased P-gp expression and
treatment outcome in AML patients.23 Furthermore, the data
shown here suggest that although CsA is an effective P-gpmodulating agent, it may fail to improve treatment response
to anthracyclines in some patients due to the existence of drug
Energy-dependence of daunorubicin uptake in AML
DW Hedley et al
transporters that are relatively insensitive to CsA. A prospective clinical study to determine the prognostic significance of
KCN-sensitive daunorubicin accumulation and detailed molecular characterization of this phenomenon are currently
under way in our institutions.
Acknowledgements
We wish to thank Dr Susan Cole, Queen’s University, Kingston, for supplying the cDNA probe for MRP, and Cynthia
Fisher and Sarah Childs for critically reading the manuscript.
This work was supported by grants from the National Cancer
Institute of Canada and the Leukemia Research Fund of Canada. Clinical material was supplied by the core of the Leukemia Program, which is supported by a Terry Fox program project grant.
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