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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4086-4095
RAPID COMMUNICATION
Altered Multidrug Resistance Phenotype Caused by
Anthracycline Analogues and Cytosine Arabinoside in Myeloid
Leukemia
By
Xiu F. Hu,
Alison Slater,
Phillip Kantharidis,
Danny Rischin,
Surender Juneja,
Ralph Rossi,
Grace Lee,
John D. Parkin, and
John
R. Zalcberg
From Trescowthick Laboratory, Peter MacCallum Cancer Institute,
Melbourne, Australia; and the Division of Pathology, Austin & Repatriation Medical Centre, Melbourne, Australia.
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ABSTRACT |
The expression of P-glycoprotein (Pgp) is often increased in acute
myeloid leukemia (AML). However, little is known of the regulation of
Pgp expression by cytotoxics in AML. We examined whether Pgp expression
and function in leukemic blasts was altered after a short exposure to
cytotoxics. Blasts were isolated from 19 patients with AML (15 patients) or chronic myeloid leukemia in blastic transformation
(BT-CML, 4 patients). Pgp expression and function were
analyzed by flow cytometric analysis of MRK 16 binding and Rhodamine
123 retention, respectively. At equitoxic concentrations, ex vivo
exposure for 16 hours to the anthracyclines epirubicin (EPI),
daunomycin (DAU), idarubicin (IDA), or MX2 or the nucleoside analogue
cytosine arabinoside (AraC) differentially upregulated MDR1/Pgp
expression in Pgp-negative and Pgp-positive blast cells. In
Pgp-negative blasts, all four anthracyclines and AraC significantly
increased Pgp expression (P = .01) and Pgp function
(P = .03). In contrast, MX2, DAU, and AraC were the most potent in inducing Pgp expression and function in Pgp positive blasts
(P < .05). A good correlation between increased Pgp
expression and function was observed in Pgp-negative (r = .90, P = .0001) and Pgp-positive blasts (r = .77, P = .0002). This increase in Pgp expression and function was
inhibited by the addition of 1 µmol/L PSC 833 to blast cells at the
time of their exposure to these cytotoxics. In 1 patient with AML, an
increase in Pgp levels was observed in vivo at 4 and 16 hours after the
administration of standard chemotherapy with DAU/AraC. Upregulation of
Pgp expression was also demonstrated ex vivo in blasts harvested from
this patient before the commencement of treatment. In 3 other cases (1 patient with AML and 2 with BT-CML) in which blasts were Pgp negative at the time of initial clinical presentation, serial samples at 1 to 5 months after chemotherapy showed the presence of Pgp-positive blasts.
All 3 patients had refractory disease. Interestingly, in all 3 cases,
upregulation of Pgp by cytotoxics was demonstrated ex vivo in blasts
harvested at the time of presentation. These data suggest that
upregulation of the MDR1 gene may represent a normal response of
leukemic cells to cytotoxic stress and may contribute to clinical drug resistance.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
MULTIDRUG RESISTANCE (MDR) is a common
obstacle to successful chemotherapy in acute myeloid leukemia
(AML).1 Patients often relapse with unresponsive disease
after an initial response to treatment with cytotoxic
drugs.2 The most common form of drug resistance in relapsed
acute leukemia is due to the overexpression of P-glycoprotein (Pgp), a
member of the ATP-binding cassette (ABC) superfamily of transporter
proteins.3 Pgp encoded by the MDR1 gene4 is
believed to function as an energy-dependent, efflux pump resulting in a
decrease in intracellular drug concentrations to sublethal levels.
There appears to be a direct correlation between expression of the MDR1
gene de novo and outcome in this disease.5,6
Other transport proteins thought to contribute to drug resistance are
the multidrug resistance-associated protein (MRP) and lung resistance
protein (LRP). Both are also expressed in AML.7 However,
the exact role and function of these proteins in AML are still
unclear.8,9 A recent study has demonstrated that MDR1/Pgp
rather than MRP or LRP expression was of prognostic value in AML and
that only MRP function was an independent prognostic factor.10
The regulation of Pgp expression is not well understood. MDR1
expression has been shown to be rapidly inducible in human cell lines
in response to a variety of stresses, including heat shock, arsenite,
or differentiating agents.11,12 The mechanisms underlying the effect of cytotoxic drugs on MDR1 gene expression and, in turn, the
MDR phenotype remain poorly understood.
The acquisition of Pgp-mediated drug resistance during chemotherapy is
usually thought to be due to the selection of drug-resistant cells.13,14 However, in previous studies in a human MDR
cell line, we demonstrated that a rapid upregulation of the MDR1 gene can occur after the exposure of cells to anthracyclines15
and its analogues.16 The rapid increase in MDR1 gene
expression after exposure to cytotoxics is strongly supported by
earlier reporter gene studies, demonstrating that the MDR1 promoter is activated by anticancer agents in human17 and rodent cell
lines.18,19 Chaudhary and Roninson20 also
demonstrated small changes in Pgp levels in a number of human cell
lines after exposure to a number of cytotoxics. Understanding the
underlying mechanisms by which cytotoxics upregulate drug resistance
genes is important, especially if this phenomenon can be prevented by
cyclosporin A (CyA) and its analogue PSC 833.21
Although studied in cell lines, the effect of cytotoxic agents on Pgp
expression in primary human cells after a short exposure to cytotoxic
agents has not been determined. The present study was designed to
investigate whether the ex vivo exposure of leukemic blasts to the
classical anthracyclines, epirubicin (EPI) and daunomycin (DAU), two
new lipid soluble anthracycline analogues idarubicin (IDA) and a new
morpholino-anthracycline MX2, or cytosine arabinoside (AraC) was able
to upregulate Pgp expression and, hence, drug resistance in AML or
chronic myeloid leukemia (CML) in blast crisis (BT-CML).
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MATERIALS AND METHODS |
Materials.
EPI, IDA, doxorubicin (DOX), and verapamil (Vp) were obtained
commercially from Pharmacia & Upjohn Pty LTD (New South Wales, Australia). DAU and AraC were purchased from David Bull Laboratories (Melbourne, Australia). MX2 was a gift from Kirin Brewery
Co Ltd (Tokyo, Japan). Vp was dissolved in 0.9% saline
solution. PSC 833 was obtained from Sandoz Pharma Ltd (Basel,
Swizerland) and initially dissolved in absolute alcohol before being
diluted in RPMI 1640 to give a stock solution of 0.5 mg/mL (the final
ethanol concentration was 35%).
The monoclonal antibody (MoAb) to Pgp (MRK 16) was generously provided
by Dr Takashi Tsuruo (Division of Experimental Chemotherapy, Japanese
Foundation for Cancer Research, Tokyo, Japan). A
fluorescein-labeled F(ab)2 fragment of sheep antimouse IgG
was purchased from Silenus Laboratories (Melbourne, Australia).
Rhodamine 123 (Rh123) and hydroxystilbamidine methanosulfonate
(Fluoro-Gold) were purchased from Molecular Probe (St Louis, MO).
Cell culture.
A variant human T-cell leukemia MDR cell line, CEM/A7R, was used as a
model for induction of MDR1 gene expression.15,16 This line
was derived from a classical MDR cell line CEM/A7 selected for low
level DOX resistance by stepwise selection of the parental line
CCRF-CEM cultured in increasing concentrations of DOX.22 The resistant line CEM/A7 was maintained in conditioned medium containing 0.07 µg/mL of DOX. The variant line (now stable for more
than 5 years) was established by culturing the CEM/A7 cells in the
absence of DOX before being subcloned and designated as the CEM/A7R
line. This line was not exposed to DOX or other Pgp substrates except
in the specific experiments detailed below. At the time of these
experiments, all lines were mycoplasma free based on the Mycoplasma T.C
Rapid kit (GEN-PROBE, Inc, San Diego, CA).
Clinical sample collection.
Peripheral blood (PB; 16 patients) or bone marrow aspirates (BM; 3 patients) were collected in EDTA or heparinized tubes from 19 patients
(15 AML and 4 BT-CML) before treatment and were diluted 1:1 with
phosphate-buffered saline (PBS) without Ca2+ or
Mg2+. Mononuclear cells were prepared by Ficoll-Hypaque
density gradient centrifugation (Pharmacia Biotech AB, Uppsala, Sweden)
according to the manufacturer's recommendations. Interface cells were
washed twice with RPMI 1640 medium (GIBCO Labs, Grand Island,
NY); resuspended in 10 mL culture medium RPMI 1640 medium;
supplemented with 10% fetal calf serum (FCS; Trace Biosciences Pty
Ltd, Melbourne, Australia), gentamicin (80 µg/mL), minocycline (1 µg/mL), HEPES (20 mmol/L), sodium bicarbonate (0.21%), glutamine
(0.8 mmol/L), 0.1% (1 µmol/L) sodium pyruvate, and 1%
nonessential amino acids; and incubated for at least 4 hours before
drug treatment. In all cases, cell viability was greater than 95% by
trypan blue exclusion and more than 75% of cells were blasts on
microscopy of cytospin specimens. When not used immediately, the
mononuclear cells were frozen in 40% RPMI 1640, 50% FCS, and 10%
dimethyl sulphoxide (DMSO) at  70°C and stored in liquid
nitrogen. The viability of frozen cells was always checked using the
trypan blue exclusion technique after thawing and was typically greater
than 95%.
Drug treatment of isolated blasts and culture conditions.
To measure the effect of drug treatment, leukemic blasts were suspended
in 10 mL of culture medium at a concentration 0.5 to 1 × 106/mL. Cells were incubated in the presence or absence of
100 ng/mL IDA, 100 ng/mL MX2, or 200 ng/mL EPI for 4 hours.
Alternatively, cells were incubated with or without 20 ng/mL IDA, 50 ng/mL MX2, 100 ng/mL EPI, 80 ng/mL DAU, or 10 ng/mL AraC for 16 hours
in the continuous presence or absence of 1 µmol/L PSC 833. The number of viable cells was determined by the use of trypan blue before and
after drug treatment. For the patients studied, no statistically significant differences were observed in cell viability in the samples
exposed to drug compared with control samples during this short time
period. Nonviable cells were excluded from flow cytometric analysis by
Fluoro-Gold, a viability stain suitable for multi-color analysis,
because it does not interfere with the emission spectrum of Rh123 or
fluorescein isothiocyanate (FITC).23
Flow cytometric analysis of Pgp expression.
MRK 16, an MoAb to an external epitope of Pgp, was used in a flow
cytometric assay to measure Pgp expression. Cells were collected and
washed 3 times in medium containing 10% FCS. MRK 16 (10 µg/mL) was
added to cells at room temperature (RT) for 20 minutes. A nonspecific
murine MoAb (IgG2a; Becton Dickinson, Sydney,
Australia) was used as the isotype control. After an
additional 3 washes, cell pellets were resuspended in the same volume
of PBS containing 10 µL of a 1:10 dilution of a
fluorescein-conjugated F(ab')2 fragment of sheep
antimouse IgG antibody (Silenus Laboratories) for 20 minutes at room
temperature in the dark. Cells were washed once again (3×) and
fluorescence was analyzed on a FACScan flow cytometer (Becton
Dickinson, Sydney, Australia) with the forward scatter (FSC) and side
scatter (SSC) gate set around the blast population and using a FSC
versus fluorogold dot plot to gate out dead cells. For each sample,
10,000 events were collected. The Lysys II software was used to analyze
data. Pgp levels were expressed as the ratio of the arithmetic mean of
the fluorescence of MRK 16 versus the IgG2a control
(refereed as Pgp ratio) in accordance with the consensus recomendations
of Marie et al.24,25 Under the conditions used, the ratio
of mean channel fluorescence of MRK16 versus control IgG2a
for CCRF-CEM ranged from 0.78 to 1.09 (0.94 ± 0.09). In the
positive control CEM/A7R line, Pgp ratios were around 2 (1.92 ± 0.32). Therefore, the threshold for Pgp negative status in blasts isolated from patients with AML or BT-CML was artificially defined as a
MCF ratio  1.10. Positive Pgp upregulation by cytotoxic treatment was
defined as an increase in the Pgp ratio of treated blasts to untreated
blasts of  10%. In some experiments, the positive MRK 16 staining
cells were also analyzed by the Kolmogorov-Smirov (KS)
test.10,26 These two methods accurately identify small differences in fluorescence and are useful in the detection of low
level protein expression, which frequently occurs in patient samples.26 A strong correlation was observed between the
two methods.10
Rh123 accumulation.
Rh123 accumulation was chosen as a sensitive and selective measure of
the transport function of Pgp.26,27 The assay was performed
as described by Broxterman et al.26 Briefly, blast cells
treated with or without cytotoxics in the presence or absence of the
modulator PSC 833 (1 µmol/L) were washed three times in RPMI with
10% FCS. Cells were then resuspended in 2 mL RPMI medium at a cell
concentration of 5 to 10 × 105 cell/mL and incubated
at 37°C for 30 minutes to allow recovery of metabolic activity.
Fluorescence was measured after the addition of 200 ng/mL of Rh123
(stock solution, 1 mg/mL in PBS) to the culture medium in the
presence or absence of 2 µmol/L PSC 833 or 10 µmol/L Vp.
The cells were then incubated in the dark at 37°C for 2 hours and
stained with 2 µmol/L Fluoro-Gold to determine cell viability.
Quantitation of fluorescence intensity was performed on the Becton
Dickinson FACSCAN using Lysis II software. Rh123 fluorescence was
measured through a 530 DF 30-nm filter and Fluoro-Gold fluorescence
through a 630 DF 32-nm filter. The acquisition gate was set on the FSC
versus Fluoro-Gold dot plot for live cell determination. The results
are expressed as the ratios of the mean channel fluorescence (MCF) in
the presence or absence of modulator (referred to as Rh123 ratio). An
increase in Pgp function by cytotoxic treatment was defined as a change
in the Rh123 ratio in treated compared with untreated blasts of 10%.
Statistical analysis.
Statistical comparisons of Pgp expression and function between
drug-treated groups versus controls were performed by analysis of
variance followed by Fisher's multiple comparison test. Correlations of Pgp upregulation and changes in Pgp function by cytotoxics were
evaluated by Spearman rank coefficient.
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RESULTS |
The upregulation of Pgp expression in AML blasts by anthracycline
analogues and AraC.
The two lipid soluble anthracyclines IDA and MX2; the classical
anthracyclines EPI, DAU, and AraC; and a non-Pgp substrate were used to
study the upregulation of Pgp in clinical samples. The
relative cytotoxicity of each of these drugs was previously determined
in the drug-resistant CEM/A7R and the parental, drug-sensitive line
CCRF-CEM in a 3-day growth inhibition assay.16 The
concentrations of each cytotoxic used in this study of clinical samples
corresponded to the IC50 levels in the CEM/A7R line determined in that
assay (20 ng/mL IDA, 50 ng/mL MX2, 100 ng/mL EPI, 80 ng/mL DAU, or 10 ng/mL AraC), although blasts were only exposed to these drugs for a
maximum of 16 hours in the current experiments.
To study the upregulation of Pgp and whether it related to the initial
Pgp status before drug treatment, leukemic blasts were categorised as
Pgp-negative (10 patients) or Pgp-positive (9 patients) based on Pgp
expression in the flow cytometric assay
(Tables 1 and
2, respectively). Initial Pgp ratios in
untreated cells ranged from 0.75 to 1.08 (0.98 ± 0.03) for
Pgp-negative blasts and 1.17 to 2.22 (1.44 ± 0.09) for Pgp-positive
blasts.
At equitoxic concentrations, ex vivo exposure for 16 hours to the
anthracyclines EPI, DAU, IDA, or MX2 or the nucleoside analogue AraC
differentially upregulated MDR1/Pgp expression in Pgp-negative and
Pgp-positive blast cells. In Pgp-negative blasts, all 4 anthracyclines and AraC significantly increased Pgp expression (P = .01, Table 1). Although every patient was not tested with every drug, all Pgp-negative blasts that were treated with MX2, EPI, or DAU displayed a
definite upregulation of Pgp levels (Table 1). In contrast, only 80%
of blast samples displayed increased Pgp expression after IDA or AraC
treatment (Table 1). In each case, the upregulation of Pgp expression
was accompanied by a significant increase in Pgp function (P = .03), as measured by a decrease in Rh123 accumulation that was
reversible in the presence of 2 µmol/L PSC 833, which was used in
this instance as a modulator of Pgp function (ie, only added to cells
at the same time as Rh123).26 There was a strong
correlation between Pgp upregulation and increased function after
exposure to cytotoxics, as shown in Fig 1A
(r = .90, P = .0001).
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| Fig 1.
(A) Correlation between Pgp upregulation and increase in
Pgp function in Pgp-negative blasts obtained from 10 patients. The data
points represent 32 independent drug treatments for which Pgp
expression and function measuments were performed as described in Table
1. Pgp expression or function was expressed as the ratio of the
arithmetic mean of fluorescence (MCF) of MRK 16 versus the
IgG2a control or Rh123 fluorescence in the presence or
absence of 2 µmol/L PSC 833, respectively, as described in Materials
and Methods. (B) Correlation between Pgp upregulation and increase in
Pgp function in Pgp-positive blasts obtained from 9 patients. The data
points represent 22 independent drug treatments for which Pgp
expression and function measuments were performed as described in Table
2. Pgp expression or function was expressed as the ratio of the MCF of
MRK 16 versus the IgG2a control or Rh123 fluorescence in
the presence or absence of 2 µmol/L PSC 833 respectively as described
in Materials and Methods. ( ) Pgp ratio/Rh 123 ratio.
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Upregulation of Pgp was also observed in the majority of Pgp-positive
blast samples. As with Pgp-negative blasts, MX2 upregulated Pgp in 8 of
9 Pgp-positive blasts. Of the 8 Pgp-positive blast samples tested with
EPI, 7 responded by upregulating Pgp expression. These 7 blast samples
were also exposed to IDA, but only 4 responded by upregulating Pgp
expression. The same 4 samples also displayed Pgp upregulation in
response to treatment with AraC or DAU. The upregulation of Pgp that
resulted from exposure to MX2, DAU, or AraC was significant, as was the
associated change in Pgp function (P < .05). From
the group of Pgp-positive blast samples, only 1 patient sample did not
show Pgp upregulation in response to any of the drugs tested. The
strong correlation between increased Pgp expression and function is
shown in Fig 1B (r = .77, P = .0002).
Inhibition of Pgp upregulation in leukemic blasts by PSC 833.
The ability of PSC 833 to inhibit the upregulation Pgp by cytotoxics
was also examined in leukemic blasts. These data are summarized in
Table 3. Upregulation of Pgp by all 5 cytotoxics was almost totally inhibited by the exposure of cells to 1 µmol/L PSC 833 at the same time as cytotoxics were added to the blast samples (Fig 2A and Table 3).
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| Fig 2.
(A) Upregulation of Pgp expression measured by flow
cytometric analysis using MRK 16 binding (solid histogram) compared
with an IgG2a control (open histogram) in Pgp-negative
leukemic blasts obtained from a single patient after 16 hours of
treatment with 20 ng/mL IDA, 50 ng/mL MX2, and 100 ng/mL EPI. The
blasts were isolated from a patient with BT-CML. The inhibitory effect
of 1 µmol/L PSC 833 on the upregulation of Pgp is also shown. Pgp
levels were expressed as the ratio of the MCF of MRK 16 versus the
IgG2a control as described in Materials and Methods. This
ratio (R) is indicated in each case. (B) Flow cytometric analysis of
Pgp function based on Rh123 accumulation in the absence (solid
histogram) or presence of 2 µmol/L PSC 833 (open histogram) in blasts
from the same patient treated as described in (A). Pgp function was
expressed as the ratio of MCF in the presence or absence of 2 µmol/L
PSC 833 as described in Materials and Methods. This ratio (R) is
indicated in each case.
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Semiquantitative reverse transcriptase-polymerase chain reaction
(RT-PCR) was used to examine the changes in MDR1 mRNA
after drug treatment in the presence or absence of 1 µmol/L of PSC
833 in 3 patient samples. The results confirmed those obtained in the
flow cytometry experiments (data not shown).
The inhibition of Pgp upregulation by PSC 833 was accompanied by
corresponding changes in Rh123 accumulation (Fig 2B). Pgp was
upregulated to different levels after 16 hours of exposure to equitoxic
concentrations of each drug, correlating with a dramatic increase in
Pgp function. This was seen as a decrease in Rh123 accumulation in
drug-treated cells. Not only was upregulation of Pgp by cytotoxics
inhibited by the presence of PSC 833, but the change in Rh123
accumulation was also prevented. An example of such an experiment is
shown in Fig 2B. Similar results were obtained for other patient
samples (data not shown).
The Pgp phenotype of AML blasts changes during induction
chemotherapy.
In blasts from a patient with AML, the change in Pgp expression in vivo
was monitored during the administration of DAU/AraC induction
chemotherapy. Blast cells were isolated from this patient before (0 hour) and at 4 and 16 hours after chemotherapy administration. Pgp
expression was significantly increased at 4 hours and further increased
at 16 hours after the onset of chemotherapy (P = .006; Fig 3A). The increase in Pgp expression was
accompanied by a significant increase in Pgp function at both time
points but was more obvious at 16 hours than at 4 hours (P = .003; Fig 3B). As recomended,30 KS analysis was also
applied to the interpretation of Pgp upregulation in this case (Fig
3A). This confirmed that the number of Pgp-positive cells significantly
increased from 1.23% ± 0.3% at 0 hour to 6.8% ± 1.0% at 4 hours and to 11.2% ± 0.8% at 16 hours after chemotherapy (P = .001).
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| Fig 3.
(A) Flow cytometric analysis of Pgp expression using MRK
16 binding (solid histogram) compared with an IgG2a control
(open histogram) in Pgp-negative blasts from a patient with AML
undergoing chemotherapy (in vivo) and after ex vivo experiments. Pgp
expression was expressed as the ratio of the MCF of MRK 16 versus the
IgG2a (R) as described in Materials and Methods. The
analysis of samples was performed before treatment (0 h), at 4 hours,
and at 16 hours of DAU/AraC treatment. In the ex vivo experiments,
cells were exposed for 16 hours to medium alone, to 100 ng/mL DAU in
the absence or presence of 1 µmol/L PSC 833, or to 10 ng/mL AraC in
the absence or presence of 1 µmol/L PSC 833. (B) Flow cytometric
analysis of Pgp function based on Rh123 accumulation in blasts from the
same patient as shown in (A) at 0, 4, and 16 hours after DAU/AraC
combination chemotherapy (in vivo) and in blasts treated with the same
drugs ex vivo. Pgp function was determined by flow cytometric analysis
using Rh123 accumulation in the absence (solid histogram) or the
presence of 2 µmol/L PSC 833 (open histogram). Pgp function was
expressed as the ratio of MCF in the presence or absence of 2 µmol/L
PSC 833 as described in Materials and Methods. This ratio (R) is
indicated in each case.
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Upregulation of Pgp and corresponding changes in Pgp function in blasts
(Fig 3A and B) from the same patient were demonstrated ex vivo by 16 hours of exposure of blast cells to 100 ng/mL DAU or 10 ng/mL AraC
harvested before the initiation of chemotherapy. The induction of Pgp
was also prevented by the presence of 1 µmol/L PSC 833 (Fig 3A).
Sequential change in Pgp status in blasts after chemotherapy.
Sequential samples from three patients with Pgp-negative blasts at the
time of clinical presentation (1 AML and 2-BT-CML) were available for
the purpose of this analysis (Table 4). Pgp expression was upregulated twofold to fourfold when blasts from these
patients were exposed ex vivo for 16 hours to equicytotoxic concentrations of 20 ng/mL IDA, 50 ng/mL MX2, or 100 ng/mL EPI. All 3 patients had Pgp-negative blasts initially (Pgp ratio of 0.75, 0.87, and 1.00; Table 4), but when retested 1, 3, and 5 months after
chemotherapy, the blasts were found to be Pgp-positive (Pgp ratio of
1.19, 1.51, and 1.76, respectively; Table 4). When a KS analysis was
applied to the initial and subsequent samples, the number of
Pgp-positive cells had increased from negative (0% to
2.3%) to 12.8%, 18.87%, and 22.1% in patients no. 1, 7, and 8, respectively. All 3 patients were refractory to treatment and died of progressive disease.
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Table 4.
Pgp Upregulation Ex Vivo Before Treatment Correlated
With the Subsequent Pgp Changes In Vivo After Chemotherapy
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DISCUSSION |
Flow cytometry analysis of Pgp expression using the MoAb MRK 16 and
changes in the accumulation of Rh123 were used in this study of blast
cells from patients with AML or BT-CML to examine Pgp upregulation. As
previously recommended,10,24-26 changes in Pgp levels
induced by cytotoxics were determined by comparing the ratios of the
MCF for the specific antibody MRK 16 over the isotope control; changes
in Pgp function were tested by comparing the MCF of Rh123 (in the
presence or absence of 2 µmol/L PSC 833).26
Using these methods, we have demonstrated the upregulation of Pgp in
leukemic blasts from patients with AML and BT-CML. At equitoxic
concentrations, ex vivo exposure to anthracyclines (EPI or DAU),
anthracycline analogues (IDA or MX2), and AraC upregulated Pgp
expression in both Pgp-negative and Pgp-positive blasts. After 16 hours
of exposure, all 4 anthracyclines as well as AraC upregulated Pgp in
Pgp-negative blasts, whereas DAU, MX2, and AraC appeared to be the most
potent in upregulating Pgp in Pgp-positive blasts (Tables 1 and 2).
There was a good correlation between Pgp upregulation and the change in
Pgp function as measured by the accumulation of Rh123 in both the
Pgp-negative and Pgp-positive blasts (Fig 1A and B).
We had previously demonstrated that upregulation of Pgp by
anthracyclines in the CEM/A7R cells was inhibited by the presence of 1 µmol/L PSC 833.21 In leukemic blasts we have now
demonstrated that the upregulation of Pgp by anthracyclines and the
non-Pgp substrate AraC was also preventable by the addition of 1 µmol/L PSC 833 (Table 3 and Fig 2A). This inhibition of Pgp
upregulation correlated with the change in Pgp function (Fig 2B). To
date, our study is the first to show that the MDR phenotype of leukemic blasts changes after a short (16 hours) exposure to cytotoxics. Upregulation of MDR1 mRNA by cytotoxic agents in acute leukemia was
also confirmed by semiquantitative RT-PCR analysis in blasts from 3 patients with AML (data not shown). The increase in MDR1 mRNA was
inhibited by the presence of 1 µmol/L PSC 833.
More direct evidence of the involvement of cytotoxics in the
regulation of Pgp expression was observed in the study of leukemic blasts from a patient undergoing chemotherapy. Pgp expression and
function were significantly increased in blasts harvested after 4 and
16 hours of exposure to DAU/AraC chemotherapy (Fig 3). Upregulation of
Pgp by DAU/AraC was also demonstrated ex vivo in blasts isolated from
the same patient before the initiation of chemotherapy (Fig 3). The
induction of Pgp by both DAU and AraC ex vivo was inhibited by the
addition of 1 µmol/L PSC 833 (Fig 3A). These data suggest that the
MDR phenotype may alter within 4 to 16 hours of the onset of
chemotherapy. How this finding impacts on clinical drug resistance
during tumor progression is not clear. However, the rapid upregulation
of Pgp expression provides evidence that the ultimate expression of the
MDR phenotype may be a direct consequence of the exposure of cells to
chemotherapeutic agents. Our observations are supported by a recent
report of a sustained increase in the drug-resistant phenotype after
chemotherapy (using a variety of cytotoxics) in neuroblastoma
cells.28 The level of drug resistance progressively
increased with the intensity of chemotherapy treatment.28
Extensive studies have provided quite good evidence that Pgp
overexpression is relatively common in primary, treatment refractory and relapsed AML.5,6 However, only a few studies have
reported that Pgp levels increase after chemotherapy.29
This was confirmed in this study in 3 patients (1 patient with AML and
2 with BT-CML) in which blasts were Pgp-negative by flow cytometry at
the time of initial clinical presentation. Pgp expression was
upregulated ex vivo by anthracyclines and AraC in blasts isolated
before the initiation of chemotherapy. Serial samples at 1 to 5 months
after chemotherapy showed the presence of Pgp-positive blasts (Table 4). All 3 patients were refractory to chemotherapy and died of progressive disease.
The mechanism by which the drugs used in this study upregulate Pgp
expression is unknown. Currently available evidence suggests that there
are at least two levels of control of MDR1 gene transcription. DNA
methylation most probably defines the first level of control. This was
originally described by Kantharidis et al30 in tumor cell
lines and a small number of chronic lymphocytic leukemia (CLL) samples and later confirmed by Nakayama et
al31 in a study of AML samples. Both studies described the
inverse correlation between methylation of the MDR1 promoter and
transcription and hence suggested that methylation may act as an on-off
switch for transcription of the MDR1 gene. The second level of control
occurs via trans-acting factors that mediate the cellular response to various stimuli and stresses. This level of control is exemplified by
the many studies that have demonstrated activation of MDR1 promoter
activity by a variety of stressful stimuli,32 including cytotoxics.17
The fact that induction can occur rapidly (4 to 16 hours) in response
to drugs that are pumped by Pgp (EPI and DAU) and others that are not
(IDA, MX2, and AraC) strongly suggests that upregulation involves a
nonspecific increase in the transcription of the MDR1 gene. The
proximal promoter of the human MDR1 gene may be directly activated by
some cytotoxic drugs17 as well as many other
factors.32 However, these findings have been controversial
with respect to their relevance in the clinical context, because the
endogenous promoter does not always behave in an analogous manner to
the transfected promoter.33,34 Both anthracycline analogues
IDA and MX2 are thought to be poor substrates for Pgp-mediated
transport due to their highly lipophilic properties, diffusing rapidly
through the cell membrane to bind to DNA.35,36 The change
in Pgp levels that resulted from exposure to these anthracycline
analogues (IDA and MX2) and the unrelated cytotoxic AraC suggests that
upregulation of the MDR1 gene may represent a normal response of
leukemic cells to cytotoxic stress that may, in turn, contribute to
clinical drug resistance. Although these cytotoxics can have a number
of effects in cells, recent findings have demonstrated that a c-jun NH2-terminal protein kinase (JNK), a member of the
mitogen-activated protein kinase family, is activated by a variety of
stressful stimuli, including exposure to cytotoxic agents, ultimately
resulting in c-jun (a key component of the AP-1 site binding factor)
phosphorylation.37 The nuclear translocation and increased
binding activity of YB-1 in response to cytotoxics has been shown to
affect Pgp expression.38 JNK is known to effect the nuclear
translocation of other factors,39 but whether it has the
same effect on YB-1 is not known.
Several studies have demonstrated that exposure to cytotoxics results
in ceramide generation,40 which, in turn, activates a
number of downstream targets, including c-raf kinase,41
protein kinase C (PKC),40 JNK,42
and the fas signaling pathway.43 Several of these targets
are known to effect expression of Pgp. For example, Cornwell and
Smith44 demonstrated activation of MDR1 promoter activity
by a signal transduction pathway involving c-raf kinase. Kim et
al45 also observed increased MDR1 expression in cell lines
after transfection with c-raf kinase.
The exact role of PKC on the Pgp-mediated MDR phenotype remains
unclear. PKC-mediated Pgp phosphorylation does not appear to correlate
with altered Pgp drug transport function.46-48 PKC isozyme is overexpressed in DOX-selected MDR cell
lines.49-51 However, increased PKC activity correlated
with MDR1 and MRP overexpression in AML,52 ovarian
carcinoma,53 and breast cancer.54 Uchiumi et
al55 demonstrated that the MDR1 promoter is activated by exposure to the cytotoxic 5-fluorourocil and this activation was blocked by the PKC inhibitor H7. Chaudhary and Roninson20
also demonstrated that the upregulation of Pgp expression by cytotoxics in a number of cell lines was blocked by the PKC inhibitors H-7 and
staurosporine. Differences in expression, substrate specificity and
activator requirements suggest that PKC isoenzymes may have distinct
roles in different signaling pathways.56 The identification of PKC cooverexpression with MDR1 in clinical samples indicates that
the isozyme PKC may play a key regulatory role in the upregulation of the MDR1 gene in response to chemotherapy.
AraC has been previously shown to activate PKC and induce both c-iun
and c-fos that make up the AP-1 transcription factor.57,58 AraC also activates JNK activity in leukemic cells59 and
the p44/42 mitogen-activated protein kinase (MAPK) that leads to
phosphorylation of serine residues in the amino-terminal
transactivation domain of c-jun.60 Thus, the upregulation
of Pgp expression by AraC may potentially be due to the combined
activation of PKC, MAPK, and JNK in myeloid leukemia blasts via the
AP-1 transcription factor.
The mechanism underlying the effect of CyA and PSC 833 on Pgp
upregulation is not known. CyA is known to bind to immunophilin A and
inhibit calcineurin in T cells and to prevent T-cell receptor translocation to the nucleus,61 ultimately causing modest
decreases in the activity of AP-3 and NF- B and marked decreases in
the activity of AP-1 and NFAT.62 CyA prevents the
dephosphorylation of NFAT, thereby preventing its translocation to the
nucleus.62 This function of CyA is likely to be important
in other cell types. JNK has been shown to bind to NFAT4 and
phospholate it at two sites, thereby preventing its translocation to
the nucleus but it also potentiates the activity of the other NFAT
isoforms through AP-1 activation.39 It is of interest that
the MDR1 promoter has binding sites for both AP-1 and AP-3, but a role
for NFAT in Pgp expression is unknown. Because the JNK pathway enhances AP-1 activity in response to cytotoxic agents, it is possible that CyA
neutralizes this activity such that induction of Pgp and other genes is
prevented. Which of these pathways and factors are involved in the
induction and inhibition response of the MDR1 gene observed in leukemic
blasts is the focus of further work in our laboratory.
| |
FOOTNOTES |
Submitted February 2, 1999; accepted March 31, 1999.
Supported in part by the Anti-Cancer Council of Victoria, the Sir
Edward Dunlop Foundation for Medical Research, and the Department of
Veterans Affairs, Canberra.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to John R. Zalcberg, MB, BS, PhD,
FRACP, Director, Division of Haematology and Medical
Oncology, Peter MacCallum Cancer Institute, Locked Bag 1, A'
Beckett Street, Melbourne, Victoria, Australia, 3000; e-mail:
zalcberg{at}petermac.unimelb.edu.au.
| |
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ABSTRACT
INTRODUCTION