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Blood, Vol. 95 No. 9 (May 1), 2000:
pp. 2897-2904
NEOPLASIA
P-glycoprotein plays a drug-efflux-independent role in augmenting
cell survival in acute myeloblastic leukemia and is associated with
modulation of a sphingomyelin-ceramide apoptotic pathway
Monica Pallis and
Nigel Russell
From the Division of Haematology, School of Clinical and Laboratory
Sciences, University of Nottingham, and the Nottingham City Hospital,
Nottingham, UK.
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Abstract |
P-glycoprotein (pgp), which is the product of the MDR1
(multidrug resistance-1) gene, has an established role as
a mediator of cytotoxic drug resistance in acute myeloid leukemia
(AML). To study the role of pgp in mediating apoptosis resistance in AML cells deprived of serum and growth factors, apoptosis was quantified by flow cytometry using uptake of the dye
7-amino-actinomycin D (7-AAD) alongside low forward scatter. In
pgp+ve primary AML samples, there was a significant increase in
apoptosis in the presence of the pgp-specific antibody UIC2 (mean
increase: 58%; range: 11%-95%; P < .05). Likewise,
apoptosis in growth factor-deprived TF1 cells cultured for 30 hours
increased 2.5-fold in the presence of 25 µg/mL UIC2. The pgp reversal
agent PSC-833 (1 µmol/L) augmented in vitro apoptosis by a median of
52% in pgp+ve patient samples and to a comparable degree in 6 pgp ve samples. To determine whether the sphingomyelin-ceramide
(SM-ceramide) pathway of apoptosis occurs in AML blasts in response to
cytotoxic drugs, cells were incubated with daunorubicin at the
patient-specific IC30 (the concentration of daunorubicin
that caused apoptotic cell death in 30% of cells) in the presence of
the ceramide synthase inhibitor fumonisin B1, which inhibited apoptosis
by 18%-81% (median: 40%). Exogenous SM failed to augment apoptosis
induced by growth factor withdrawal in pgp+ve TF1 cells and
was significantly more effective at augmenting apoptosis in pgpve
patient blasts (median increase in cell death: 33%; range: 19%-88%)
than in pgp+ve samples (median: 7%; range: 0%-27%;
P = .028). Cellular accumulation of exogenous SM was
associated with apoptosis and also occurred in nonapoptotic patient
cells treated with PSC-833. However, this effect was not seen following
treatment with the UIC2 antibody. These results indicate that pgp is
able to exert a protective effect on AML cell viability and that this
is associated with a reduced effect of exogenous SM on apoptosis. The
pgp reversal agent PSC-833 acts, at least in part, by a pgp independent
mechanism to alter SM distribution and to augment apoptosis induced in
AML cells by serum and growth factor withdrawal.
(Blood. 2000;95:2897-2904)
© 2000 by The American Society of Hematology.
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Introduction |
P-glycoprotein (pgp) is the product of the
MDR-1 (multidrug resistance-1) gene and is responsible for
transporting a variety of amphiphilic substances across plasma
membranes.1 The term MDR-1 was conferred on the gene
because of the ability of its product pgp to efflux a broad range of
chemotherapeutic drugs such as anthracyclines and
etoposide.2 Expression of pgp on leukemic cells has been
measured in several series of patients with AML and has been
established as a major indicator of poor prognosis.3-15
Until recently, only 1 important role for pgp in tumor biology, namely
its ability to efflux drugs, has been elucidated. However, a further
role has now been demonstrated in a T-cell leukemia model: anti-fas,
UV, and drugs induce caspase-mediated apoptosis, which is
inhibited by pgp.16,17 In Chinese hamster ovary
fibroblasts, pgp expression was found to delay apoptosis induced by
growth factor withdrawal.18
The role of the sphingomyelin-ceramide (SM-ceramide) pathway has not
previously been reported in primary AML cells, although a role for both
SM hydrolysis19 and ceramide
synthesis20 has been reported in cell line studies using
daunorubicin. This pathway may be important in pgp-augmented cell
survival because pgp has been reported to transport phospholipids
across cell membranes.21 It has been hypothesized that pgp
may act as a primary antiapoptotic molecule by reducing the pool of
plasma membrane SM that can be hydrolyzed to ceramide.22
Most plasma membrane SM is expressed in the outer leaflet, but a small
signaling pool of internalized SM has been identified.23,24
Sphingomyelin can be hydrolyzed to ceramide by neutral sphingomyelinase
in response to exposure to proapoptotic stimuli such as tumor necrosis
factor-alpha (TNF- ) or daunorubicin.25 The pgp
modulator PSC-833 has been found to enhance SM redistribution from the
outer to the inner plasma membrane and subsequent apoptosis of KG1a
cells treated with TNF-.22 It is not known whether
PSC-833 was acting as a specific pgp reversal agent in this context.
The primary purpose of the current investigation was to establish
whether there is a role for pgp in the apoptosis of primary leukemic
cells independent of its role in mediating multidrug resistance. For
this purpose we have used the pgp blocking antibody UIC226
as well as the pgp reversal agent PSC-833. We also established the
relevance of a SM-ceramide apoptotic pathway in the chemoresistance of
leukemic cells using the specific ceramide synthase inhibitor fumonisin
B1 (FB1).20 Finally, to investigate whether there may be a
role for pgp-mediated SM compartmentalization in the apoptosis
resistance of pgp+ve leukemic cells, we added exogenous fluorescent SM
(C6-NBD-SM) to pgp+ve and pgpve leukemic cells and
compared its effect on augmenting apoptosis in the 2 cell types as well
as the effect of PSC-833 on cellular SM distribution.
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Materials and methods |
Reagents
The following products were used in this study: MRK-16 anti-pgp
(Kamiya, Seattle, WA); 15D3 anti-pgp (Becton Dickinson, Cowley, England); control and secondary antibodies for pgp analysis (Dako, High
Wycombe, England); rhodamine 123 (R123), 7-amino-actinomycin D (7-AAD),
the annexin V apoptosis detection kit, propidium iodide, FB1,
L-glutamine, and antibiotics (Sigma Aldrich, Poole, England); daunorubicin (Cerubidin, Rhone-Poulenc Rorer, West Malling, UK); C6-NBD-SM (Molecular Probes, Cambridge Bioscience,
Cambridge, England); and granulocyte-macrophage-colony stimulating
factor (GM-CSF) (Leucomax, Novartis, Basel, Switzerland).
Fumonisin B1 was dissolved in sterile water and stored at 4°C.
PSC-833 (gift from Novartis) was stored at 1 mmol/L in ethanol. A 1 mmol/L stock solution of C6-NBDSM in ethanol was stored at 20°C. UIC2 anti-pgp blocking antibody (Immunotech, Beckman
Coulter, High Wycombe, England) was stored in 200 µg/mL aliquots at
20°C, as were preservative-free control IgG2a antibodies
(Calbiochem, Nottingham, England). Fetal calf serum (FCS) (First Link,
Wolverhampton, England) was heat inactivated at 56°C for 60 minutes.
Cells
U937 and TF1 leukemic cell lines were grown in RPMI
1640 with 10% FCS, 2 mmol/L L-glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin. The medium of the growth factor dependent cell line TF1 was supplemented with 200 units/mL GM-CSF. Experiments were performed on cells in log phase. Fresh or cryopreserved blood or
bone marrow samples from untreated patients with AML were used in this
study. As several assays were to be performed on the same samples, all
samples selected were from patients with high blast counts; 9 samples
were collected at presentation, and the remaining 2 were from relapsed
patients (RF and GM in Table 1). The
viability of thawed cells was assessed using trypan blue dye exclusion, and only samples with more than 90% thawed cell viability were used.
Determination of MDR status
The MDR status of patient blasts was determined by 3 previously
described methods.15 First, pgp was measured using an
unconjugated MRK-16 antibody and a fluorescein
isothiocyanate-conjugated (FITC-conjugated) second layer. In 1 case
there was high nonspecific binding of the control antibody, and the
phycoerythrin-conjugated (PE-conjugated) 15D3 antibody gave a clear
result. This antibody was also used to confirm protein results in 2 protein-negative, R123 modulation-positive samples. Pgp substrate
efflux modulation by PSC-833 was determined in an accumulation assay
using R123. Thawed or freshly isolated patient blasts were rested in
culture medium containing 20% FCS for 60-90 minutes. They were then
pelleted; resuspended at 106/mL; and incubated in medium
containing 10% FCS at 37°C, in duplicate, with 200 ng/mL R123,
with or without 2 µmol/L PSC-833. Control tubes with R123 incubated
at 4°C were also set up. Following a 75-minute incubation, the
cells were pelleted at 4°C, rinsed, and resuspended. They were
analyzed using a FACScan or FACSCalibur flow cytometer (Becton
Dickinson) as previously described.15 The mean fluorescence
intensity (MFI) of each sample in the FL1 channel was recorded. Results
were expressed as (MFI with PSC-833-cold control MFI)/(MFI with
diluent control-cold control MFI). Third, results were validated using
the standardized daunorubicin uptake assay. Rested leukemic blasts at
106/mL and carboxylate microspheres (Polysciences) were
incubated in duplicate for 75 minutes with 2 × 10 6
mol/L daunorubicin at 37°C. Control cells were also set up with daunorubicin at 4°C. Cells and beads were rinsed and analyzed as
described.15 Results were expressed as (cell MFI-control cell MFI)/bead peak fluorescence.
Cell culture
Thawed or fresh mononuclear cell preparations from patients with AML
were rested for 60-90 minutes in culture medium consisting of RPMI 1640 with 20% FCS and 1% L-glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin. The cells were then counted, rinsed in 20 mL
RPMI, and resuspended at 5 × 105/mL in culture medium
with only 1% FCS. Only samples with a post-rest viability greater than
90% were used. For the C6-NBD-SM assay, aliquots were
incubated for 30 minutes at 37°C at 2 × 106/mL with
4 µmol/L C6-NBD-SM in 1% FCS, pelleted, and resuspended in 1% FCS. Preliminary studies of pgp+ve cells had indicated that 30 minutes of preincubation with C6-NBD-SM was sufficient for saturation, and that 30 minute fluorescence at a concentration of 4 µmol/L C6-NBD-SM was approximately 1 log greater than
autofluorescence in pgp+ve samples.
For chemosensitivity assays, cells were cultured for 48 hours in medium
containing 10% FCS and dilutions of daunorubicin ranging from 0.01-1 µg/mL. The IC30 (ie, the concentration of daunorubicin that caused apoptotic cell death in 30% of cells) was determined for
each patient and used for a follow-up assay in which patient cells were
cultured with daunorubicin at the IC30 with and without 50 µmol/L FB1. Apoptotic cell death was quantified using 7-AAD as
detailed below.
Determination of apoptotic cell death
7-AAD was diluted in PBS as a 50 µg/mL stock solution, which was
stored at 4°C. This was mixed with cultured cells to give a final
concentration of 12.5 µg/mL. Cultures were incubated for 20 minutes
in the dark and analyzed using flow cytometry (Cellquest software,
Becton Dickinson). Analysis was performed as illustrated by
Philpott27 and Schmid28 to select cells
undergoing apoptotic cell death, which was represented by 7-AAD-high
cells with low forward scatter. The apoptotic nature of the cell death
induced by growth factor withdrawal was examined in additional studies using MGM-stained cytospins for cells displaying
morphological characteristics of apoptosis, ie, condensed nuclear
chromatin and membrane blebbing, and by quantitative analysis using the annexin V/propidium iodide kit (Sigma Aldrich) according to
manufacturer's instructions.
Statistical analysis
Statistical analyses were performed (software; SPSS,
Chicago, IL). The differences in response to anti-pgp and control
antibodies were analyzed using the Wilcoxon signed rank test.
Differences in responses between pgp+ve and pgpve groups were
analyzed using the Mann-Whitney U test.
The D value used to measure pgp is calculated in the Kolmogorov-Smirnov
test by the flow cytometer's Cellquest software (Becton Dickinson) and
is a measure, on a scale of 0 to 1, of the difference in distribution
between 2 fluorescence curves. The D value is used to calculate the
difference between test and control fluorescence when that
difference is likely to be small.15,29 Values of less
than 0.1 were considered to be negative.
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Results |
MDR status of patient cells and cell lines
P-glycoprotein protein and function are measured
routinely on AML samples in our laboratory. Out of 11 samples, 5 protein positive (pgp D values: 0.15-0.47; mean: 0.30) and 6 protein
negative samples were selected for further study (Table 1). PSC-833
increased R123 uptake more than twofold in 4/5 pgp+ve samples, but its
effect was less marked in pgpve samples. Daunorubicin uptake was
also characterized in these patients (Table 1). A previous study has shown that a daunorubicin cell to bead ratio of less than 2.1 is
associated with MDR mediated by pgp, lung resistance protein (lrp), or
multidrug resistance-associated protein (mrp).15
Daunorubicin uptake was found to be low in 4/5 pgp+ve samples and 2/6
pgpve samples. TF1 cells are strongly pgp+ve
(D = 0.93 ± 0.03), with a high PSC-833 modulation
(5.0 ± 0.47), whereas U937 cells are pgpve phenotypically
and have a low modulation ratio (1.12 ± 0.01).
Apoptosis in patient cells and TF1s augmented by
UIC2 anti-pgp
To examine a role for pgp in apoptosis that could be separated from
its role in drug uptake, a growth and serum factor withdrawal system
was chosen, as this induces considerable apoptosis in most AML patient
samples as well as in the pgp+ve growth factor-dependent TF1 cell line
within 48 hours. Apoptotic cell death was quantified by flow cytometry
using uptake of the dye 7-AAD in addition to low forward scatter. The
quantitative use of 7-AAD to measure apoptosis was validated by
comparison with the measurement of apoptotic cells identified by
exposure of phosphatidyl serine using annexin V (Figure
1). A close association was found, as has
also been noted by others.30 Cultured leukemic patient
cells do not undergo cell death at the same rate, and moreover they undergo considerable secondary necrosis, such that many of the nonviable cells observed after overnight incubation are necrotic rather
than apoptotic. Figure 1 illustrates that the propidium iodide-positive (necrotic) cells are also annexin V positive, strongly
indicating that necrosis was secondary to apoptosis. The apoptotic
nature of cell death induced by growth factor withdrawal was examined
in additional studies through MGM-stained cytospins, which showed cells
displaying morphological characteristics of apoptosis, ie, condensed
nuclear chromatin and membrane blebbing (data not shown).
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| Fig 1.
Comparison of 7-AAD and annexin V for the
quantitation of apoptotic cell death.
AML patient cells were incubated overnight in 1% serum without growth
factors and without (A, B) or with (C, D) 1 µmol/L PSC-833, the pgp
reversal agent. We added 12.5 µg/mL 7-AAD to tubes (A) and (C). Tubes
(B) and (D) were rinsed and stained with annexinV FITC and propidium
iodide. Flow cytometer dot-plots illustrate nonviable cells: high 7-AAD
in FL3, with decreased forward scatter in plots (A) and (C), positive
annexin V in FL1 on plots (B) and (D). Secondary necrosis is also seen
in plots (B) and (D) on the FL2 axis (propidium iodide incorporation).
The percentage of cells determined by each method to be undergoing or
to have undergone apoptosis is indicated on the plots.
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In pgp+ve primary AML samples cultured overnight in 1% FCS without
growth factors, there was an increase in apoptotic cell death in all
samples assayed in the presence of the pgp-specific antibody UIC2
compared with the isotype- and concentration-matched control antibody, with a mean increase of 58% (range: 11%-95%; P < .05 using the Wilcoxon signed rank test) (Table
2 and Figure 2). TF1 cells were also cultured in 1% FCS
without growth factors in the presence of UIC2 and control IgG2a
antibodies. At 30 hours, apoptosis had increased 2.5-fold compared with
that of cells cultured with isotype- and concentration-matched control
antibodies (Figure 3). In the presence of
growth factors, UIC2-induced apoptosis was less than 1% in all
experiments, demonstrating that the antibody was not toxic. As an
additional negative control, apoptosis was also measured in U937 cells
deprived of growth factors overnight in the presence of UIC2 or
control IgG2a. We did not observe UIC2-dependent apoptosis.
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Table 2.
The effect of UIC2 anti-pgp antibody and IgG2a control
antibody on apoptotic cell death in pgp+ve leukemic samples
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| Fig 2.
The effect of UIC2 on blast survival in pgp+ve and
pgpve patient samples.
Primary AML cells were cultured in 1% FCS with UIC2 anti-pgp and
isotype- and concentration-matched control antibodies. Flow cytometer
dot-plots illustrate nonviable cells (high 7-AAD in FL3, decreased
forward scatter) in (A) IgG2a and (B) UIC2-treated cells
(patient RF). Numbers on plots are the percentages of nonviable
cells.
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| Fig 3.
UIC2 augments apoptosis in TF1 cells deprived of growth
factors.
Unfilled squares represent cells treated with 25 µg/mL UIC2. Filled
squares represent IgG2a control-treated cells. Standard errors for 1 experiment are illustrated. Data points were plotted after the
subtraction of control values from cells grown in the presence of
GM-CSF with or without the appropriate antibody. Similar results were
obtained in 2 further experiments.
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The pgp reversal agent PSC-833 also augments in vitro apoptosis in
TF1s and patient cells cultured without growth factors
The effect of the pgp inhibitor PSC-833 on growth factor withdrawal
in TF1 and primary AML blasts was also determined. TF1 cells were
cultured with 0.5- and 1-µmol/L concentrations of PSC-833, which were
found to enhance the cell death induced by growth factor withdrawal in
a dose-dependent manner (Figure 4). In the
presence of growth factors, PSC-833-induced apoptosis was less than
2% in all experiments, which demonstrates minimal toxicity for this compound.
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| Fig 4.
PSC-833 augments apoptosis in TF1 cells deprived of
growth factors.
Percentage loss of TF1 viability after culture for 23 and 30 hours in
1% FCS without growth factors in the absence (filled circles) or
presence of 0.5 µmol/L (filled diamonds) or 1 µmol/L
(unfilled circles) PSC-833 (the mean plus or minus SE of 4 experiments). Data points were plotted after the subtraction of control
values from cells grown in the presence of GM-CSF with or without the
appropriate dose of PSC-833.
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A pgp independent role for PSC-833 in interleukin-1 (IL-1) secretion
and leukemic blast survival has recently been postulated in
AML.31 To determine whether the apoptotic effect of PSC-833 on growth factor withdrawn cells was pgp specific, pgp+ve and pgpve patient cells were cultured in the presence of PSC-833, which was found to enhance cell death in all samples tested including 6 pgpve samples (Figures 1 and 5). At
1 µmol PSC-833, in pgpve samples, the increase was 6%-350%,
and the median was 53%; in pgp+ve samples, the increase was
13%-204%, and the median was 52%. There was no significant
difference between groups using the Mann-Whitney U test. These
experiments indicated that the enhancement of cell death induced by
growth and serum factor withdrawal by PSC-833 is not pgp specific.
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| Fig 5.
The effect of PSC-833 on blast survival in pgp+ve and
pgpve patient samples.
Leukemic patient sample viability after treatment with PSC-833 in
18-hour culture in 1% FCS without growth factors. Thick lines
represent responses of pgp+ve blasts, and thin lines represent
pgpve blasts.
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Inhibition of daunorubicin-induced apoptosis by the ceramide
synthase inhibitor FB1
The role of the SM-ceramide pathway in apoptosis is controversial.
The controversy largely surrounds the lack of specificity of the lipid
reagents used and the methodology for measuring ceramide up-regulation.32 Before investigating the possible role of
pgp in SM distribution, it was thought advisable to establish a role for the SM-ceramide pathway in the apoptosis of primary AML cells. The
natural fungal product FB1 is structurally similar to the ceramide
precursor sphinganine and inhibits ceramide synthase activity, thus
inhibiting de novo ceramide synthesis.20 To the best of our knowledge, the specific inhibitory role of FB1 in ceramide
synthesis has not been challenged, and this compound has previously
been used to demonstrate a role for the ceramide pathway in the
daunorubicin-induced apoptosis of U937 cells.20 We
therefore cultured primary leukemic samples with and without daunorubicin and FB1. The presence of FB1 was found to reduce daunorubicin-specific apoptosis by a median of 40% (range: 18%-81%) in the 7 samples studied (Table 3). This
implicates a direct role for ceramide in daunorubicin-induced apoptosis
in AML cells.
SM in the apoptosis of pgp+ve and pgpve leukemic
cells
The low bioavailability of plasma membrane inner leaflet SM has been
postulated as a limiting factor in TNF-induced apoptosis in a pgp+ve
leukemic cell model.22 To expand this hypothesis we
constructed models using a fluorescent SM, C6-NBD-SM, to
label pgp+ve and pgpve leukemic cell membranes. If the
hypothesis proposed by Bezombes et al22 were applicable, we
would expect to observe that C6-NBD-SM, transported to the
inner leaflet of stressed pgpve cells, would augment apoptosis;
whereas in pgp+ve cells, the exogenous SM would be largely confined to
the outer leaflet, and apoptosis would not be enhanced. We measured the
effects of exogenous C6-NBD-SM on apoptosis. Preliminary
experiments using flow cytometry to measure cell fluorescence enabled
us to observe that C6-NBD-SM uptake reached a plateau
within 30 minutes at 4 µmol/L in pgp+ve cells (data not shown), and
this loading schedule was adopted in subsequent experiments. An
experiment was performed on pgpve U937 cells to determine
whether C6-NBD-SM induces apoptosis or whether it augments
a preinduced apoptotic pathway. U937 cells survived for several days in
1% FCS; we therefore used daunorubicin (rather than serum and growth
factor withdrawal) to induce apoptosis in these cells. Minimal
apoptosis was induced by C6-NBD-SM alone in the absence of
daunorubicin (Figure 6A). However, in
combination with daunorubicin, C6-NBD-SM enhanced
apoptosis. In contrast, C6-NBD-SM was ineffective at
augmenting apoptosis induced by growth factor withdrawal in pgp+ve TF1
cells (Figure 6B).
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| Fig 6.
The effect of SM on apoptosis in pgp+ve and pgpve
cell lines.
(A) U937 cells were cultured in 10% FCS for 3 hours in the presence of
increasing doses of daunorubicin. The cells were then rinsed, incubated
for 30 minutes with or without C6-NBD-SM in 1% FCS,
pelleted, and cultured for a further 18 hours. In the absence of
daunorubicin, C6-NBD-SM made no difference to U937
viability, demonstrating that SM was not inherently toxic at this dose.
However, daunorubicin-induced apoptosis was augmented in a
dose-dependent manner by C6-NBD-SM. Error bars equal the
standard error of the mean (SEM) of 3 experiments. (B) The lack of
effect of C6-NBD-SM on apoptosis induced by growth factor
withdrawal in pgp+ve TF1 cells is demonstrated. TF1 cells were loaded
with C6-NBD-SM and cultured with and without GM-CSF. With
growth factor withdrawal, 16% of cells were nonviable by 30 hours, but
C6-NBD-SM did not enhance apoptosis. Error bars equal SEM
of 3 experiments.
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The effect of C6-NBD-SM on the in vitro apoptosis of
primary leukemic cells was then studied. Following a 30-minute
incubation with C6-NBD-SM, blast samples were pelleted and
incubated for a further 18 hours at 37°C in 1% FCS. In 6 pgpve cases, increases of 19%-88% in cell death, median 33%,
were observed, whereas in the 5 pgp+ve cases, increases of 0%-27%,
median 7%, were noted (Table 4 and Figure
7). The Mann-Whitney U test
demonstrated the difference between groups to be significant at
P = .028.
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Table 4.
The effect of C6-NBD-SM on augmenting
apoptosis induced by growth factor and serum withdrawal in primary
leukemic blasts
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| Fig 7.
The effect of SM on apoptosis in pgp+ve and pgpve
patient samples.
Flow cytometer dot-plots illustrating nonviable cells (high 7-AAD in
FL3, decreased forward scatter) in a pgpve sample (AC) without
(A) and with (B) C6-NBD-SM and in a pgp+ve sample (GO)
without (C) and with (D) C6-NBD-SM.
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Accumulation of exogenous SM by AML blasts is enhanced by
PSC-833
When exogenous phospholipids are incubated with cells in the
presence of protein, the lipid retained in the membrane is affected by
both uptake into the cell and back-exchange from the outer leaflet into
the protein-containing medium.33 We therefore postulated that 18 hours after C6-NBD-SM loading, fluorescence in
leukemic cells would be related to the amount of SM internalized. The
studies by Bettaib et al34 documented low inner leaflet SM
content in pgp+ve KG1a cells compared with pgpve U937 cells and
subsequently determined that PSC-833 can enhance inner leaflet SM
content in the presence of an apoptotic stimulus.22
Therefore, we were interested in C6-NBD-SM fluorescence
distribution in apoptotic and nonapoptotic cells. We examined
cytofluorographs from samples that had been incubated overnight with
C6-NBD-SM in 1% FCS without growth factors (Figure
8). From these, we determined that high C6-NBD-SM fluorescence is largely associated with apoptotic
cells. (Cells with reduced forward scatter and increased side scatter have been shown to be apoptotic in several models.35,36)
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| Fig 8.
C6-NBD-SM fluorescence of leukemic blasts
following 18-hour culture.
(A) Blasts from pgpve patient LC incubated overnight with
C6-NBD-SM demonstrate intense fluorescence on the subset of
cells defined by region 5 (R5). Backgating of the (B) R4 and (C) R5
subsets demonstrated that R5 C6-NBD-SM high cells were the
smaller cells with higher side scatter, typical flow cytometric
features of apoptosis.35 The figure is a representative
plot of a consistent finding. (D) Histogram of the same sample
incubated without (filled plot) and with (unfilled plot) 1 µmol/L
PSC-833, which demonstrates an increase in C6-NBD-SM high
cells. (E, F) C6-NBD-SM-loaded blasts from pgp+ve patients
(E) GO and (F) RF demonstrate a fluorescence shift in the entire
population as well as in the fluorescence-high subset when untreated
samples (filled plots) are compared to samples treated with PSC-833
(unfilled plot, thick line). (F) The lowering effect of UIC2 (unfilled
plot, dotted line) on C6-NBD-SM fluorescence is also
shown.
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Patient cells were also treated with both C6-NBD-SM and 1 µmol/L PSC-833. C6-NBD-SM fluorescence was increased by
PSC-833 in 10/11 patient samples (all except JD). Increased
C6-NBD-SM may reflect an alteration in SM distribution
preceding apoptosis or alternatively may be nonspecifically associated
with a loss of cellular integrity in these samples. To distinguish
between these possibilities, we examined cultures from pgp+ve samples that underwent only minimal apoptosis at 18 hours. An increase in
C6-NBD-SM fluorescence was clearly distinguishable in the
viable cells of samples treated with PSC-833 (Figure 8), supporting the finding that PSC-833 alters SM distribution.22 However, the pgp blocking antibody UIC2 was also used in conjunction with
C6-NBD-SM in these assays and was found to cause a slight
fall in the C6-NBD-SM fluorescence of viable pgp+ve cells
(Figure 8). This suggests that PSC-833 is altering SM distribution, at
least in part, by a pgp independent mechanism.
| |
Discussion |
Using the pgp-specific blocking antibody UIC2, we demonstrated a
drug efflux independent role for pgp in augmenting the survival of
primary leukemic blasts and TF1 cells in vitro. A similar role in
apoptosis augmented by growth factor withdrawal was previously demonstrated in Chinese hamster ovary fibroblasts.18 When
added to the findings of Smyth16 and
Johnstone,17 who have demonstrated a role for pgp in the
fas- and UV-induced caspase-mediated apoptosis of T cell lines, it is
clear that a fundamental role for pgp in leukemic cell survival, which
is much broader than its drug efflux role, has now been established. It
is important to note in this context that only blocking antibodies can
be considered as totally pgp specific. UIC2 is a well-documented
antibody that recognizes an extracellular portion of pgp but not of the
MDR 2/3 gene product.26,37,38 UIC2 reactivity is increased
in the presence of pgp substrate drugs or in experiments in which
adenosine 5'-triphosphate (ATP) is directly
depleted, and it has therefore been suggested that the inhibitory
function of the antibody may involve the trapping of a transient
conformation of pgp associated with ATP hydrolysis.39 Many
pharmaceutical agents that inhibit pgp function, including PSC-833,
have broader roles,31 and in this study we have shown that
PSC-833 enhances cell death when growth and survival factors are
withdrawn from pgpve as well as pgp+ve patient samples. A direct
cytotoxic effect of PSC-833, independent of initial blast pgp status,
has also been noted in an in vitro AML blast chemosensitivity assay.40
Apart from pgp's function as a drug efflux pump, it has been suggested
that pgp can transport phospholipids across cell
membranes.21 In view of this, a potential role for pgp in
reducing the bioavailability of SM has been postulated.22
To determine whether reduced SM is relevant to the induction of
apoptosis in AML blasts exposed to cytotoxic drugs, we initially used
the ceramide synthase inhibitor FB1, which blocks the de novo
synthesis of ceramide. In this system FB1 was found to reduce
daunorubicin-specific apoptosis. In this respect our findings agree
with those of Bose et al,20 who, like ourselves, cultured
cells continuously with daunorubicin. In contrast, Jaffrezou et
al19 found evidence for SM hydrolysis but not for de
novo ceramide synthesis in the daunorubicin-induced apoptosis of
U937 cells after a 1-hour pulse of daunorubicin. As ceramide is both a
precursor and a breakdown product of SM,41,42 the precise role of de novo ceramide synthesis vis-à-vis
SM hydrolysis as contributors to apoptosis will be hard to establish
and may partly depend on an experimental schedule. Furthermore, a
direct drug-independent role for PSC-833 in enhancing ceramide
synthesis has been shown in pgp+ve and pgpve epidermoid
carcinoma cells.43 It also has been recently demonstrated
that PSC-833 interacts with daunorubicin to induce SM hydrolysis and
ceramide synthesis in drug-resistant HL60 leukemic cells.44
As further evidence of a role for the SM-ceramide pathway in the
apoptosis of AML cells, we have shown that exogenous
fluorescence-labeled SM enhances loss of viability induced by
daunorubicin in the pgpve U937 cell line. Although SM was
not toxic when used without drugs, the availability of
exogenous SM enhanced daunorubicin-induced apoptosis. The availability
of SM is therefore shown to be a factor limiting the degree
of apoptosis in AML cells in response to cytotoxic drug
exposure. The evidence for a proapoptotic role for SM is further
supported by the demonstration that SM augments apoptosis in
pgpve patient blasts. In contrast, SM failed to enhance
apoptosis in the pgp+ve TF1 cell line and had a significantly decreased effect on pgp+ve patient blasts compared with that seen in
the pgpve cases.
Many of the models used to study pgp have involved the selection of
drug-resistant sublines in vitro, which tend to express pgp at much
higher levels than those found in unselected leukemic cell
lines.45 The unselected TF1 cell line has low pgp
expression compared to other pgp+ve AML cell lines.45
However, even this cell line expresses more pgp than any of the
100-plus clinical AML samples studied in our laboratory (data not
shown). For this reason we consider that it is important to use patient
data to help understand whether an in vitro pgp model is likely to be pathophysiologically relevant in AML. However, it is also
important, when evaluating the data which we have presented, to realize
that a pgp+ve patient sample may contain a mixture of pgp+ve
and pgpve blasts. Hence the differences in SM metabolism between
pgp+ve and pgpve cells may be even more clear-cut than our
patient data reveals.
The choice of a fluorescence-labeled SM for this study enabled us to
observe the uptake and retention of this compound on a single-cell
basis. We found that the addition of PSC-833 enhanced the SM
fluorescence in viable cells, compatible with the finding of Bezombes
et al22 that PSC-833 enhanced the internalization of SM,
potentially providing an internal pool of hydrolyzable SM to
up-regulate ceramide-mediated apoptosis. However, we were unable to
establish that PSC-833 was acting specifically on pgp in this context
because the pgp-specific UIC2 antibody, while inducing a similar degree
of apoptosis at 18 hours, did not increase C6-NBD-SM
fluorescence. Cyclosporin A, which is closely related to PSC-833, is
known to have a membrane fluidizing effect,46 which may
account for an altered, pgp-independent distribution of phospholipids
in the plasma membrane. In this context, cyclosporin A was also found
to enhance C6-NBD-SM fluorescence in our system (data not shown).
The central finding of this study has been the demonstration that when
pgp is blocked, apoptosis induced by growth factor withdrawal in acute
myeloid leukemic cells is enhanced. We have also demonstrated for the
first time that the SM-ceramide pathway is involved in cytotoxic
drug-induced apoptosis in primary AML blasts. Furthermore we have shown
that exogenous SM enhances apoptosis induced by growth factor
withdrawal in pgpve AML blasts, but that pgp+ve cells are
protected from SM enhancement of apoptosis. PSC-833 was found not only
to enhance apoptosis but also to increase cellular SM accumulation. The
fact that PSC-833 enhances cell death and SM internalization in both
pgp+ve and pgpve cases is of particular clinical relevance, as
this agent is currently undergoing clinical evaluation, and these
findings suggest that it may be of benefit to a broader range of
patients than just those with pgp+ve blasts.
The question of how pgp exerts its antiapoptotic effects remains open.
Pgp-mediated alterations of the electrochemical gradient across the
plasma membrane are well documented and may be relevant in this
context.47,48 Additionally, a role for pgp has been documented in the transport of several membrane
lipids.21,49,50 There is also a reported role for
pgp in cytokine export.51 Which, if any, of these
mechanisms is relevant to the survival of leukemic clones is currently
a major focus of research in our laboratory.
| |
Footnotes |
Submitted May 12, 1999; accepted December 20, 1999.
Reprints: Monica Pallis, Academic Haematology, Clinical
Sciences Building, Nottingham City Hospital, Nottingham NG5 1PB, UK;
e-mail: m.pallis{at}nottingham.ac.uk.
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.
| |
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Abstract
Introduction