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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 1075-1085
P-Glycoprotein Protects Leukemia Cells Against Caspase-Dependent,
but not Caspase-Independent, Cell Death
By
Ricky W. Johnstone,
Erika Cretney, and
Mark J. Smyth
From The Austin Research Institute, Austin Hospital, Heidelberg,
Victoria, Australia.
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ABSTRACT |
A major problem with treating patients with cancer by traditional
chemotherapeutic regimes is that their tumors often develop a multidrug
resistant (MDR) phenotype and subsequently become insensitive to
a range of different chemotoxic drugs. One cause of MDR is
overexpression of the drug-effluxing protein, P-glycoprotein.It is now apparent that P-glycoprotein may also possess a more generic antiapoptotic function that protects P-glycoprotein-expressing cancer
cells and normal cells from cell death. Herein we show that cells
induced to express P-glycoprotein either by drug selection or by
retroviral gene transduction with MDR1 cDNA are resistant to cell death
induced by a wide range of death stimuli, such as FasL, tumor necrosis
factor (TNF), and ultraviolet (UV) irradiation, that activate the
caspase apoptotic cascade.However, P-glycoprotein-expressing cells were not resistant to caspase-independent cell death mediated by
pore-forming proteins and granzyme B.MDR
P-glycoprotein-expressing cells were made sensitive to
caspase-dependent apoptosis by the addition of anti-P-glycoprotein
antibodies or verapamil, a pharmacological inhibitor of P-glycoprotein
function. Clonogenic assays showed that P-glycoprotein confers
long-term resistance to caspase-dependent apoptotic stimuli but not to
caspase-independent cell death stimuli. This study has confirmed a
potential novel physiological function for P-glycoprotein and it now
remains to dissect the molecular mechanisms involved in the inhibition
of capsase-dependent cell death by P-glycoprotein.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
MULTIDRUG RESISTANCE (MDR) is a
well-defined phenomenon of cross-resistance of mammalian cells to a
number of anticancer agents following exposure to one such
drug.1 A diverse range of agents involved in MDR include
alkaloid compounds and bacterial and fungal antibiotics, such as
anthracyclines and etoposide. An accepted mechanism of MDR is a reduced
cellular accumulation and an altered subcellular distribution of
cytotoxic drugs. In many instances, this is mediated by increased
expression at the cell surface of the MDR1 gene product, P-glycoprotein
(P-gp), a 170-kD energy-dependent efflux pump.2,3
Transfection studies have clearly shown that P-gp is responsible for
MDR.4 MDR-mediated by P-gp is thought to be primarily due
to reduced intracellular concentrations of drug and failure of the drug
to reach its target.
A number of models have been proposed to explain the mechanism of
action of P-gp in MDR. The traditional model for P-gp function was one
where P-gp acts as a "drug pump" to export drugs out of a cell
against a concentration gradient. This has been further expanded to the
"flippase" model, which attempts to explain how P-gp can remove a
range of structurally diverse drugs without an apparent substrate
specificity.5,6 However, given that there is not always a
linear correlation between extent of drug efflux and resistance to cell
death, it appears that P-gp may also prevent cell death by some
additional mechanism or that other cellular proteins also influence
resistance to certain death stimuli. In addition to its drug efflux
activity, P-gp has been suggested to function as an ion
channel,7 and expression of P-gp may result in
alkalinization of the cytosol due to an increase in pHi and
altered membrane potential (Vm).8
P-gp is normally expressed in the epithelial cells of kidney, liver,
pancreas, and intestine; and in capillary endothelia in brain and
testes, consistent with its role in the export of xenotoxins out
of exposed epithelial cells; or across the blood-testes or blood-brain
barrier.9-11 In addition, P-gp is expressed on CD34+ hematopoietic progenitor cells and on normal human
natural killer (NK) cells and CD8+ T cells.12
The functional significance of the regulated expression of P-gp on
specific hematopoietic cells has yet to be established. However,
immunological or developmental defects in mice with functional deletion
of the Mdr1a and Mdr1b genes have not been reported.13,14
The molecular events leading to programmed cell death or apoptosis
involve an increasingly well-defined cascade of proteolytic cleavage
events. The key cell death proteins are the cysteine-aspases (caspases), which are initially expressed as inactive zymogens and are
activated by proteolytic processing to produce the active enzyme.15,16 It is now clear that cell death initiated by a range of stimuli, including FasL,17 tumor necrosis factor
(TNF),18 ultraviolet (UV) irradiation,19,20
granzyme B (GzB),21,22 and chemotherapeutic
drugs,20,23 function by activating this central death
cascade. Cytotoxic T lymphocytes (CTL) can kill target cells by two
independent mechanisms: the granule exocytosis mechanism using the
granule proteins such as perforin (pfp) and GzB, and the FasL-Fas
pathway.21,22 Cell death due to membrane and cytosolic
pertubations by pfp/GzB occurs in the absence of activation of the
caspase pathway, whereas nuclear damage requires caspase
activation.24 Thus, while most apoptotic stimuli have been
shown to activate caspases, pfp/GzB have been shown to additionally induce cell death in a caspase-independent manner.
Chinese hamster ovary cells expressing P-gp are more resistant to
apoptosis induced by serum deprivation,23 and expression of
P-gp confers resistance to Fas-mediated apoptosis. This resistance to
Fas-mediated apoptosis can be reversed by using antibodies that bind
the extracellular domains of P-gp or verapamil, a pharmacological inhibitor of P-gp function.25 Expression of P-gp results in decreased production of active caspase 3, a key effector caspase in the
apoptosis cascade upon Fas ligation. Inhibition of P-gp function
restores caspase 3 activation upon crosslinking of cell-surface Fas.25 These preliminary studies were performed in only one pair of drug-selected cell lines but suggested P-gp could inhibit cell
death mediated strictly via the caspase pathway. Herein we have
extended this work by using P-gp expressing cells produced by
retrovirus transduction of MDR1 cDNA and several drug-selected cell
lines. In addition, we have tested a wide variety of caspase-dependent or -independent death stimuli to determine the protective effect of
functional P-gp on the short-term and long-term survival of cells. P-gp
conferred protection against all forms of caspase-dependent apoptosis
studied, but cells were sensitive to caspase-independent cell death
stimuli regardless of their P-gp expression.
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MATERIALS AND METHODS |
Cell culture.
The acute T-cell leukemia cell line, CEM-CCRF, its doxorubicin
(DOX)-selected and resistant P-gp expressing line
CEM-A7+, and various hybrids of CCRF and A7+
expressing high (IC10) or negligible (2H6) levels of P-gp have been
previously described.26 The T-cell line 12D7 and production of 12D7-MDR1 cells by transduction with a retrovirus containing the
MDR1 gene was described previously.27 All cells were grown in RPMI medium supplemented with 10% (vol/vol) fetal calf serum (FCS),
2 mmol/L glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin
(GIBCO, Grand Island, NY).
Cytotoxicity assays.
Effector cells and various soluble death stimuli were assessed by
51Cr release or [125] iododeoxyuridine
(125IUdR) release assays as described.25 DOX,
etoposide (VP16), and vincristine (VIN) were obtained from Dr Phillip
Kantharidis, Peter MacCallum Cancer Institute, East Mebourne,
Australia. Dexamethasone (Sigma, St Louis, MO) and CH-11 (anti-human
Fas IgM, Upstate Biotech Inc, Lake Placid, NY) were purchased.
Recombinant soluble human TNF was kindly provided (Peprotech, Rocky
Hill, NJ). Rat pfp and human GzB were purified as previously
described28 and were used in combination as
described.25 The spontaneous release of 51Cr or
125IUdR was determined by incubating the target cells with
medium alone (or in the presence of anti-P-gp monoclonal antibody
[MoAb], verapamil, or caspase inhibitor where applicable). It should
be noted at the concentrations used, inhibitors alone did not cause release, nor did they affect the long-term survival of cell lines. The
maximum release was determined by adding sodium dodecyl sulfate (SDS)
to a final concentration of 5%. The percent specific lysis was
calculated as follows: 100 × [(experimental release  spontaneous release)/(maximum release spontaneous release)].
To inhibit P-gp function, the labeled targets were preincubated for 30 minutes with MRK-16 (IgG2a MoAb, final 1 to 100 µg/mL; Kamiya
Biochemical Company, Thousand Oaks, CA), UIC2 (IgG2a MoAb, final 0.1 to
5 µg/mL; Coulter, Miami, FL), or verapamil (0.5 to 10 µmol/L; Knoll Australia, Lane Cove, Australia) before the cytotoxicity assay. Isotype
control antibodies were included where applicable. To inhibit caspase
activity, labeled target cells were preincubated for a further 30 minutes with peptidyl fluoromethyl ketones (fmk; ZFA-fmk, ZVAD-fmk;
Enzyme System Products, Dublin, CA; final 0 to 50 µmol/L).
UV irradiation.
Cells were plated at 1 × 106 cells/mL in flat-bottom
24-well plates and irradiated with UV light by placing the plate,
without a lid, directly under a UV transilluminator. Cells were removed at appropriate times and allowed to incubate at 37°C for 24 hours before apoptosis was assessed. Apoptotic cells were identified by two
separate assays. Trypan blue exclusion assays were performed by mixing
an equal volume of cells with 0.3% Trypan blue, and cells from four
fields of vision were counted. The percentage of apoptotic cells was
also determined by treating cells with propidium iodide (PI) as
described,29 and cells in the sub
G0/G1 population were detected using a FACScan
(Becton Dickinson, San Jose, CA).
Clonogenic assays.
Cells (5 µL from a stock of 4 × 104 cells/mL)
treated with various apoptotic stimuli were plated out in triplicate on
soft agar. Cells were diluted in 5 mL RPMI containing 20% (vol/vol) FCS, the supplements listed above, and 0.3% noble agar
(Difco, Detroit, MI) and plated in 60-mm dishes. Once set,
the dishes were overlaid with 2.5 mL of media, incubated at 37°C
for 12 days, and the total number of colonies per plate were counted.
Anti-P-gp immunoblotting.
CEM and 12D7 subclones (2 × 105) were lysed in 50 µL of ice-cold Nonidet P-40 lysis buffer (25 mmol/L HEPES, pH 7/250
mmol/L NaCl/2.5 mmol/L EDTA/0.1% Nonidet P-40/0.5 mmol/L DTT/2 mmol/L Pefabloc) at 4°C for 30 minutes. Insoluble material was removed by
centrifugation at 4°C. Proteins (25 µg) were separated on
SDS-10% polyacrylamide gels and electroblotted onto nylon membranes.
Blots were probed with anti-P-gp MoAb C219 (Sapphire Bioscience,
Sydney, Australia) and visualized by enhanced chemiluminescence.
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RESULTS |
Drug-selected P-gp+ve cell lines are
resistant to caspase-dependent apoptosis induced by chemotherapeutic
drugs.
It has been reported that chemotherapeutic drugs induce cell death by
activating the caspase apoptotic pathway.20,23 To confirm
this finding and to determine the effect of P-gp expression on
caspase-mediated apoptosis, we analyzed the cytotoxicity of different
drugs on matched P-gp+ve and P-gp ve cell
lines. We initially determined P-gp expression on CEM-CCRF, its
DOX-selected P-gp+ve-derived line CEM-A7+, and
two hybrids of CCRF and A7+ by Western analysis
(Fig 1, lanes 1 through 4). A7+
and IC10 express high levels of P-gp (Fig 1, lanes 2 and 4), whereas
CCRF and 2H6 are P-gpve (Fig 1, lanes 1 and 3).
These analyses were confirmed by flow cytometry using two separate
MoAbs, MRK-16 and UIC2, which bind to extracellular domains on P-gp
(data not shown). Exposure P-gpve cell lines to the
anthracycline, DOX, resulted in DNA fragmentation (Fig 2A, lanes 1, 4, 13, and 16) and
membrane lysis (Fig 2B, lanes 1, 4, 13, and 16), as shown by
125I-DNA and 51Cr release, respectively. By
contrast, cell death in P-gp+ve cell lines was
significantly decreased (Fig 2A and B, lanes 7, 10, 19, and 22). The
cell death events induced by DOX in P-gpve cells
were blocked with the generic caspase inhibitor ZVAD-fmk (Fig 2A and B,
lanes 2, 5, 14, and 17), but not with the control ZFA-fmk (Fig 2B,
lanes 3, 6, 15, and 18). Similar cell death assays using the vinca
alkaloid, VIN; the glucocorticoid, DEX; and the topoisomerase II
inhibitor, VP16, revealed that these different classes of drugs used at
multiple doses all induced both nuclear and cell membrane damage in the
P-gpve cell lines but had far reduced effects on
P-gp+ve cell lines (Table 1,
one drug dose shown). The cell death induced by these different agents
was inhibited by ZVAD-fmk but was not affected by ZFA-fmk (Table 1).
These data indicate that the two different P-gpve
cell lines tested were sensitive to different classes of
chemotherapeutic drugs, whereas P-gp+ve cell lines were
truly MDR. Despite their different modes of action, all of the drugs
were inhibited by ZVAD-fmk, indicating that they all induce apoptosis
in a caspase-dependent manner.
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| Fig 1.
Expression of P-gp on drug-selected and retrovirus
transduced cell lines. Western blot of cell lysates from drug-selected
CEM cell lines CCRF, A7+, 2H6, IC10; the T-cell line 12D7
and 12D7 transduced with a retrovirus containing full-length human MDR1
cDNA (12D7-MDR1) were probed with the anti-P-gp MoAb, C219. The
position of 170 kD P-gp is indicated by the arrow on the right.
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| Fig 2.
Drug-selected and retroviral transduced
P-gp+ve cells are resistant to DOX-mediated,
caspase-dependent DNA fragmentation and membrane lysis. Drug-selected
CEM cell lines (P-gpve CCRF, 2H6; P-gp+ve
A7+, IC10) (A, B) and parental 12D7
(P-gpve) and retrovirus transduced 12D7-MDR1
(P-gp+ve) cells (E, F) were labeled with
125IUdR and 51Cr for 1 hour, washed in growth
media, and incubated for 48 hours in 96-well plates (2 × 104 cells/well) with DOX. In some wells, cells were
preincubated for 30 minutes with 20 µmol/L ZVAD-fmk or control
ZFA-fmk inhibitor as indicated in Table 1. P-gp+ve cells
were made sensitive to DOX-mediated DNA fragmentation (C) and membrane
lysis (D) by preincubation with anti-P-gp MoAb, MRK 16 (50 µg/mL).
DNA fragmentation and membrane lysis were reflected by
125I-DNA release (A, C, E) and 51Cr release (B,
D, F), respectively. Data are calculated as the mean ± SE of
triplicate samples and are representative of a least two different
experiments.
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DOX-mediated DNA fragmentation and membrane lysis was induced in
A7+ and IC10 by inhibiting the function of P-gp with a
specific MoAb directed to an extracellular domain of P-gp (Fig 2C and
D, lanes 2 and 6). Cell death in these lines was inhibited by the
addition of ZVAD-fmk (Fig 2C and D, lanes 3 and 7), but not by ZFA-fmk (Fig 2C and D, lanes 4 and 8), showing that reversal of resistance to
chemotherapeutic drugs by P-gp rendered these cells sensitive to
caspase-dependent cell death. Similar results were observed in
A7+ and IC10 cells treated with VP16, MRK 16, ZVAD-fmk, and
ZFA-fmk (data not shown).
Cells expressing P-gp by retroviral transfer of the MDR1 gene are
resistant to caspase-dependent chemotherapeutic drugs.
To show that the protection by P-gp against caspase-dependent
apoptosis induced by different chemotherapeutic drugs is not restricted
to DOX-selected CEM cells, two other matched P-gp+ve and
P-gpve cell lines were used. The cell lines used
were the human T-cell lines, 12D7 (P-gpve), and
12D7-MDR1 (P-gp+ve; Fig 1, lanes 5 and 6). 12D7-MDR1 is a
stable cell line produced by transduction of 12D7 cells with a
retroviral construct containing the wild-type MDR1 cDNA.27
Treatment of 12D7 with DOX resulted in DNA fragmentation (Fig 2E, lane
1) and membrane lysis (Fig 2F, lane 1) that was inhibited with ZVAD-fmk
(lane 3), but not ZFA-fmk (lane 5). By contrast, 12D7-MDR1 cells were
relatively resistant to DOX-mediated cell death (Fig 2E and F, lane 7),
but were rendered sensitive to DNA and membrane damage by DOX upon addition of the anti-P-gp MoAb (Fig 2E and F, lane 8). As with the
other P-gp+ve cell lines used above, cell death induced in
12D7-MDR1 cells by drug and anti-P-gp MoAb was caspase-dependent (Fig
2E and F, lanes 10 and 12). Similar data were obtained from 12D7 and
12D7-MDR1 cells treated with VP16 (data not shown).
Cells expressing P-gp are resistant to Fas-mediated apoptosis.
Recently, we have shown that A7+ cells were resistant to
cell death induced by the Fas pathway.25 To expand these
studies on P-gp+ve cells, Fas-mediated apoptosis was
directly induced by three different methods. Cell surface Fas
expression on these cells has been assessed previously and has been
shown to be equivalent25 (and data not shown). Treatment of
P-gpve CCRF and 2H6 cells with anti-Fas MoAb to
cross-link Fas on the cell surface resulted in significant,
dose-dependent DNA fragmentation (Fig 3A
lanes 1 through 6, 13 through 18) and membrane lysis (Fig 3B lanes 1 through 6, 13 through 18) that was blocked by pretreatment of cells
with ZVAD-fmk (Fig 3A and B, lanes 2, 5, 14, and 17) but not ZFA-fmk
(Fig 3A and B, lanes 3, 6, 15, and 18). By contrast, P-gp+ve cells were relatively resistant to Fas-mediated
apoptosis (Fig 3A and B, lanes 7 through 12, 19 through 24).
Preincubation of A7+ with two different anti-P-gp MoAbs
(MRK 16 and UIC2) rendered them sensitive to apoptosis by anti-Fas MoAb
(3C and D, lanes 5 through 11). P-gp+ve 12D7-MDR1 cells
were also resistant to Fas-mediated DNA fragmentation (Fig 3E, lanes 7 through 9, 19 through 21) and membrane lysis (Fig 3F, lanes 7 thorugh
9, 19 thorugh 21), and this resistance could be reversed by the
addition of MRK 16 (Fig 3E and F, lanes 10 and 22) or UIC2 (data not
shown). Fas-mediated cell death was caspase-dependent in 12D7-MDR1
P-gp+ve cells treated with MRK 16 (Fig 3E and F, lanes 11 and 23) or UIC2 (data not shown). Treatment of P-gp+ve and
P-gpve cells with soluble, recombinant FasL also
resulted in caspase-dependent apoptosis of Pgpve
cells, whereas P-gp+ve cells were relatively resistant
(data not shown). To use a more physiological stimulator of
Fas-mediated cell killing, we employed the rat/mouse hybrid cell line
d12S, which expresses FasL on the cell surface and kilsl target cells
in a FasL-dependent manner. Incubation of cells with d12S resulted in
caspase-dependent apoptosis of P-gpve target cells
(data not shown), but did not induce similar membrane lysis or DNA
fragmentation in P-gp+ve cells.
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| Fig 3.
Drug-selected and retroviral-transduced
P-gp+ve cells are resistant to Fas-mediated,
caspase-dependent DNA fragmentation and membrane lysis. CEM cell lines
(A, B) (P-gpve CCRF, 2H6; P-gp+ve
A7+, IC10), parental 12D7 (P-gpve), and
retrovirus-transduced 12D7-MDR1 (P-gp+ve) cells (E, F)
were labeled with 125IUdR and 51Cr for 1 hour,
washed in growth media, and incubated for 16 hours in 96-well plates (2 × 104 cells/well) with anti-human Fas IgM MoAb, CH-11
(0.01 µg/mL). In some wells, cells were preincubated for 30 minutes
with 20 µmol/L ZVAD-fmk or control ZFA-fmk inhibitor and/or
anti-P-gp MoAb MRK 16 (50 µg/mL) as indicated in Table 1. CCRF and
A7+ cells (C), (D) were preincubated with the given
concentrations of anti-P-gp MRK 16 MoAb (1 to 50 µg/mL) or UIC2 MoAb
(0.1 to 5 µg/mL), then treated with 0.01 µg/mL anti-Fas MoAb for 16 hours. Data are calculated as the mean ± SE of triplicate samples and
are representative of at least two different experiments.
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P-gp confers resistance to cell death induced by UV irradiation.
Exposure of cells to UV light has been shown to induce
caspase-dependent apoptosis due to cross-linking of cell surface Fas and subsequent activation of the Fas/caspase cell death
pathway.19 We therefore tested CCRF/A7+ and
12D7/12D7-MDR1 cells for their relative sensitivity to UV irradiation.
P-gp+ve cells A7+ and 12D7-MDR1 were less
sensitive to UV irradiation-induced apoptosis than the parental
P-gpve cell lines CCRF and 12D7 as determined by
Trypan blue exclusion assays and fluorescence analysis following PI
staining (data not shown). We subsequently pretreated CCRF and
A7+ cells with anti-P-gp MoAbs (MRK 16 or UIC2) or
verapamil and then exposed the cells to UV irradiation. Resistance of
A7+ to UV irradiation was reversed by all three anti-P-gp
reagents (Fig 4A, lanes 5 through 16),
whereas treatment of CCRF with the P-gp inhibitors had no effect on
sensitivity to UV irradiation (Fig 4B, lanes 5 through 16). Viability
of P-gpve cells following UV irradiation was
increased by preincubation in ZVAD-fmk (data not shown). Similar
reversal of resistance to UV-induced cell death was seen in 12D7-MDR1
cells treated with P-gp inhibitory agents (data not shown).
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| Fig 4.
P-gp+ve cells are resistant to cell death
induced by UV irradiation. A7+ and CCRF cells, 4 × 104 cells in 3-mL media, were subjected to UV-irradiation
(2 to 10 minutes), removed from the UV source, and incubated for 24 hours. Cell viability was determined by Trypan blue exclusion and
confirmed by fluorescence analysis following staining with PI (data not
shown). In some wells, cells were preincubated with MRK 16 (50 µg/mL), UIC2 (5 µg/mL), or verapamil (5 µmol/L) for 30 minutes.
Data are calculated as the mean ± SE of quadruplicate samples and are
representative of at least two different experiments.
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Cells expressing P-gp are resistant to TNF-mediated apoptosis.
Fas is a member of the TNF receptor (TNFR) superfamily, and a common
intracellular caspase pathway is activated leading to apoptosis by Fas
or TNFR ligation.16 As TNF can also induce caspase-dependent apoptosis when added to cells expressing TNFR, we
tested for the effect of P-gp expression on TNF-mediated cell death.
P-gp+ve and P-gpve cells expressed
equivalent levels of TNFR as determined by binding assays using
radiolabeled recombinant TNF (data not shown). Treatment of
P-gpve CCRF (data not shown) and 12D7
(Fig 5A and B) cells with TNF resulted in
DNA fragmentation and membrane lysis, which was inhibited by the
addition of ZVAD-fmk (Fig 5A and B, lanes 2, 5, 14, and 17), but not
ZFA-fmk (Fig 5A and B, lanes 3, 6, 15, and 18). Both A7+
(data not shown) and 12D7-MDR1 (Figs 5A and B) were relatively resistant to TNF, however pretreatment with MRK 16 resulted in sensitivity to caspase-mediated apoptosis (Fig 5A and B, lanes 10 through 12, 22 through 24). Similar reversal of resistance of
A7+ and 12D7-MDR1 to TNF was observed using the UIC2 MoAb
or verapamil (data not shown).
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| Fig 5.
Retroviral transduced P-gp+ve cells are
resistant to TNF-mediated, caspase-dependent DNA fragmentation and
membrane lysis. Parental 12D7 (P-gpve) and
retrovirus-transduced 12D7-MDR1 (P-gp+ve) cells were
labeled with 125IUdR (A) and 51Cr (B) for 1 hour, washed in growth media, and incubated for 48 hours in 96-well
plates (2 × 104 cells/well) with recombinant soluble TNF
(0.2 and 2.0 µg/mL). In some wells, cells were preincubated for 30 minutes with 20 µmol/L ZVAD-fmk or control ZFA-fmk inhibitor
and/or anti-P-gp MoAb MRK 16 (50 µg/mL) as indicated in
Table 1. Data are calculated as the mean ± SE of triplicate samples
and are representative of at least two different experiments.
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P-gp+ve cells treated with caspase-dependent
apoptotic stimuli retain proliferative and colony-forming ability.
The results presented above are from relatively short-term assays (4 to 48 hours). To determine whether P-gp truly protected cells against
caspase-dependent apoptosis or merely delayed the apoptotic events,
colony formation assays were performed. This is a stringent in vitro
assay because it examines the ability of single cells to proliferate
and form colonies. Treatment of CCRF with anti-Fas MoAb
(Fig 6A) or purified recombinant TNF (Fig 6C) caused a significant reduction in the number of colonies that grow
in soft agar (Fig 6A and C, lanes 1 and 2). Pretreatment of CCRF cells
with ZVAD-fmk protected them against Fas- or TNF-induced cell death and
restored the number of colonies growing per plate. Pretreatment with
ZFA-fmk had no effect on Fas- or TNF-mediated cell death as measured by
colony formation (Fig 6A and C, lanes 3 through 6). By contrast,
A7+ cells were resistant to anti-Fas- and TNF-mediated
cell death as treatment of cells with these apoptotic stimuli in the
presence or absence of ZVAD-fmk or ZFA-fmk had no effect on colony
formation (Fig 6B and D, lanes 1 through 7). However, pretreatment of
A7+ cells with MRK 16 MoAb resulted in reversal of
resistance to these stimuli, and the resultant cell death was
caspase-dependent (Fig 6B and D, lanes 7 through 12).
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| Fig 6.
P-gp+ve cells treated with
caspase-dependent apoptotic stimuli retain proliferative and
colony-forming ability. CCRF (A, C) and A7+ (B, D) cells
(4 × 104 cells/mL) were treated with anti-Fas MoAb (0.01 mg/mL) (A, B) for 16 hours, or with recombinant soluble TNF (0.2 ng/mL)
(C, D) for 48 hours. Cells were assayed for colony-forming ability by
plating out 5 µL of cells in soft agar and incubating at 37°C for
12 days. In some wells, cells were preincubated for 30 minutes with 20 µmol/L ZVAD-fmk or control ZFA-fmk inhibitor and/or
anti-P-gp MoAb MRK 16 (50 µg/mL) as indicated in Table 1. Data are
calculated as the mean ± SE of triplicate plates and are
representative of at least two different experiments.
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P-gp does not protect cells against caspase-independent cell death.
CTL and natural killer (NK) cells destroy target cells via a granule
exocytosis mechanism involving pfp and GzB, two key cytolytic molecules
present in the cytoplasmic granules of these immune cells. Purified GzB
and pfp can induce both caspase-dependent nuclear and
caspase-independent cytoplasmic pertubation and membrane lysis of
target cells.22 Treatment of P-gp+ve and
P-gpve cells with sublytic concentrations of pfp in
combination with increasing amounts GzB resulted in significant DNA
fragmentation in CCRF (Fig 7A, lanes 1 through 3) and 12D7 cells (data not shown) but not of A7+
(Fig 7A, lanes 10 through 12) or 12D7-MDR1 cells (data not
shown). The DNA fragmentation in CCRF cells was caspase-dependent (Fig 7A, lanes 4 through 9), whereas cell membrane lysis of CCRF or A7+ was caspase-independent (Fig 7B, lanes 7 through 9, 16 through 18). Both A7+ (Fig 7A, lanes 19 through 21) and
12D7-MDR1 cells (data not shown) pretreated with MRK 16 were
subsequently more sensitive to DNA fragmentation induced by pfp/GzB.
There was no significant difference in long-term survival of CCRF or
A7+ exposed to GzB and pfp (data not shown). This indicates
that the caspase-independent cytoplasmic events induced by GzB/pfp, in
the absence of DNA degradation, are sufficient to induce cell death.
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| Fig 7.
Caspase-independent cell lysis is unaffected by P-gp
expression. P-gp+ve and P-gpve CEM cell
lines were labeled with 125IUdR (A, C) and 51Cr
(B, D) for 1 hour, washed in growth media, and incubated for 16 hours
in 96-well plates (2 × 104 cells/well) with a sublytic
concentration of pfp (30 U) and increasing amounts of GzB (0.2, 0.5, 2.0 µg/mg) (A, B) or with pfp alone (300 U) (C, D). In some wells,
cells were preincubated for 30 minutes with 20 µmol/L ZVAD-fmk or
control ZFA-fmk inhibitor and/or anti-P-gp MoAb MRK 16 (50 µg/mL) as indicated in Table 1. Data are calculated as the mean ± SE of triplicate samples and are representative of at least two
different experiments.
|
|
Pfp and the functionally related bacterial toxin, pneumolysin, can lyse
cells by forming pores in the target cell membrane to induce cell
death.22 Treatment of Pgp+ve and
P-gpve cells with either of two concentrations of
purified pfp (Fig 7C) or pneumolysin (data not shown) resulted in
equivalent membrane damage in all cells that were not affected by
ZVAD-fmk or ZFA-fmk (Fig 7D). Consistent with previous reports, pfp
(Fig 7C) or pneumolysin (data not shown) did not cause significant DNA
fragmentation when added to either P-gp+ve or
P-gpve cells. Similar results were seen in 12D7 and
12D7-MDR1 cells where pfp alone caused membrane lysis but not DNA
fragmentation (data not shown).
Thus, P-gp confers resistance to caspase-dependent cell death stimuli
such as Fas, TNF, and UV irradiation, but the presence of P-gp on the
cell surface does not affect cell death induced by caspase-independent
death stimuli.
| |
DISCUSSION |
P-gp, responsible for multidrug resistance of tumor cells, has
traditionally been thought to function by actively effluxing toxic
compounds from cells.1-3 We have recently shown that
drug-selected cells expressing P-gp were protected from cell death
mediated by cross-linking cell surface Fas,25 and Robinson
et al23 have shown that P-gp-expressing cells produced by
gene transfer are resistant to apoptosis induced by serum deprivation.
Because the outgrowth of cells in the presence of cytotoxic drugs may cause cellular changes other than P-gp expression, we used different paired P-gp+ve and P-gpve cell lines,
produced by either drug selection or by transfection of the MDR1 cDNA,
and inhibitors of P-gp function to show that expression of P-gp confers
resistance to apoptosis mediated by a range of cell death stimuli.
Protection by P-gp was dependent on the mode of action of the cell
death mediator. P-gp protected against all stimuli that function by
activating the caspase pathway but did not affect caspase-independent
cell death. Significantly, the anti-apoptotic function of P-gp could be
inhibited by the use of two MoAbs that bind to different extracellular
epitopes or by the pharmacological inhibitor of P-gp function,
verapamil. This protection and reversal of resistance of
P-gp+ve cells was shown in short-term 51Cr and
125I-DNA release assays and in long-term survival or
clonogenic assays. The clonogenic assays are important because they
show that functional P-gp inhibits apoptosis by caspase-dependent
stimuli and does not merely delay death in P-gp+ve cells
treated with apoptotic inducers.
Although the mechanism of anti-apoptotic action of P-gp remains to be
defined, a number of hypotheses can be put forward. It has been
suggested that P-gp may efflux large proteins such as interleukin-2
from a cell30 and thus it is possible that a key caspase or
another mediator of apoptosis is being removed from P-gp+ve
cells. We have shown previously that CCRF and A7+ cells
express equivalent amounts of intracellular caspase 3, but that the
cleavage and activation of this important molecule was inhibited in
A7+ cells when exposed to apoptotic stimuli.25
We therefore, have no evidence to suggest that caspases are effluxed in
P-gp+ve cells exposed to apoptotic stimuli, but other
mediators of apoptosis may be affected. It is possible that P-gp
affects apoptosis by altering intracellular adenosine triphosphate
(ATP) levels. ATP is a critical component for the
activation of caspase 9.31 P-gp is an ATPase, and even when
not effluxing specific drugs, has constitutive ATPase
activity.1,32,33 Thus, it seems possible that a decrease in
the intracellular ATP pool may affect caspase-mediated apoptosis.
However, verapamil, which reverses the anti-apoptotic effect of
P-gp,1,23,25 increases the ATPase activity of
P-gp,33,34 presumably further decreasing intracellular ATP
levels. Thus, we do not favor this hypothesis, although careful studies
need to be performed to determine the minimum intracellular ATP levels required for caspase 9 activation and the effect P-gp has on these levels. It has been reported that expression of P-gp is associated with
intracellular alkalinization,1,35 and an elegant study by
Hoffman and Roepe8 showed that P-gp induces a novel
Na+- and Cl-dependent pathway for
transmembrane H+ transport to bring about this change in
intracellular pH (pHi). It has been shown that apoptosis
induced by Fas ligation, UV irradiation, serum starvation, or
chemotherapeutic drugs is preceded by intracellular acidification,29,36 and that the induction of apoptotic
events such as DNA laddering can be inhibited by increasing
pHi.23 Cells that are normally sensitive to
apoptosis by Fas cross linking29 or serum
starvation23 can be made resistant to these
caspase-dependent death stimuli by elevating pHi. Thus, it
is possible that expression of P-gp alters pHi placing the
cell in a state of caspase-inactivity, and thereby rendering
P-gp+ve cells relatively resistant to multiple forms of
caspase-dependent cell death stimuli. The addition of verapamil causes
a reversal in intracellular alkalinization of P-gp+ve
cells,23 which correlates with increased sensitivity to a
range of apoptotic stimuli23,25 (and this study). We have
yet to determine what effect MoAbs MRK 16 and UIC2 have on
pHi; however, it has been shown that binding of anti-P-gp
MoAbs to extracellular P-gp epitopes causes a change in conformation of
the molecule, possibly holding it in an "inactive"
state.37
P-gp is expressed normally on epithelial cells of the gut, liver, and
kidney,38 which is consistent with its function in removing
toxins from cells. However, P-gp is also expressed on some
hematopoietic cells, including NK and T lymphocytes,12 that
can induce apoptosis of target cells via granule exocytosis (involving
pfp and granule serine proteases such as GzB)22 or Fas
ligation.39 It is not immediately obvious why NK and T
cells require a drug efflux pump, but given our data, it is possible that P-gp expression may protect immune cells against stress-induced or
bystander lysis in the hostile environment of sites of inflammation. We
have previously shown that bystander lysis by cytotoxic T lymphocytes in vitro occurs predominantly via the Fas pathway,40 and
because P-gp expression confers resistance to this form of cell death, it follows that the primary immune cells expressing P-gp could be
somewhat protected against Fas-mediated apoptosis in vivo. It has also
been shown that bystander lysis may be mediated by TNF,41
and our data clearly show that P-gp+ve cells are relatively
resistant to TNF-mediated cell death. There has been conflicting
reports showing that P-gp expression correlates with either
increased42-44 or decreased45 sensitivity of
cells to TNF-mediated apoptosis. However, these studies were
potentially flawed given that the cells used were selected only on the
basis of their drug resistance. We favor the findings that P-gp confers resistance of cells to TNF given that we can reverse resistance of
drug-selected P-gp+ve cells to TNF with anti-P-gp
antibodies, and that retrovirally transduced P-gp+ve cells
are similarly resistant to TNF. P-gp is also expressed on pluripotent
CD34+ stem cells that can proliferate and differentiate to
give rise to all of the mature hematopoietic cells of the
body.12 The physiological function of P-gp on this subset
of cells is currently unknown; however, it is conceivable that
progenitor cells would have mechanisms in place to generally protect
them against apoptotic stimuli, and it is tempting to speculate that
P-gp could help serve this purpose.
Different chemotherapeutic agents have been shown to induce the death
of susceptible cells by apoptosis,20,23,46 and this can be
inhibited by proteins such as Bcl-247 and
Bcl-xL.48 The different P-gp+ve and
P-gpve cell lines used in this study express
equivalent amounts of Bcl-2 and the intracellular levels of Bcl-2 to
not alter in response to death stimuli such as drug or Fas ligation in
the presence or absence of anti-P-gp MoAbs (Johnstone et
al, unpublished observation, July 1998). We tested a number of
cytotoxic drugs of various classes with specific intracellular targets
on different parental P-gpve cells and on
P-gp+ve, and in all cases P-gp conferred resistance to
these stimuli. The mechanisms of chemotherapy-induced,
caspase-dependent apoptosis have thus far not been delineated, and
there is some controversy as to the role of upregulated FasL on the
surface of cells treated with chemotherapeutic agents. It has been
shown that treatment of cells with chemotherapeutic drugs or DNA
damaging agents such as UV irradiation resulted in upregulated FasL
expression, causing Fas-mediated apoptosis.20,49 However,
recently it has been shown that the triggering of apoptosis by a broad
array of chemotherapeutic agents occurs in a Fas/FasL-independent
fashion but the downstream effector proteins (ie, caspases) are the
same as those activated by Fas ligation.50 In addition,
cells expressing a dominant-negative form of FADD, which is a key
adapter between receptors such as Fas and TNFR, and caspase 8 are not
protected from cell death induced by a range of apoptotic stimuli
including chemotherapeutic drugs.51 It is presently unclear
whether the presence of P-gp affects the expression and/or
function of FasL. However, we have recently shown that K562 cells,
which are resistant to apoptosis mediated by anti-Fas MoAbs, are still
sensitive to caspase-dependent cell death mediated by chemotherapeutic
agents,25 and KVIN cells (VIN selected K562) that express
P-gp can be made sensitive to a range of caspase-dependent apoptotic
stimuli by addition of anti-P-gp MoAbs or verapamil.
Although P-gp expression confers resistance to a range of different
caspase-dependent apoptotic stimuli, cell death induced by
caspase-independent mediators were not affected. Recently, a number of
molecules capable of inducing apoptosis in the presence of caspase
inhibitors have been reported, including the GD3
ganglioside52 and the adenovirus protein,
E4orf4.53 Although the molecular mechanisms leading to cell
death by these and other caspase-independent stimuli have yet to be
fully delineated, it is apparent that this mode of apoptosis-induction
may have implications in both physiological and pathological cell death.
| |
ACKNOWLEDGMENT |
We thank Drs M.M. Gottesman and C.G. Lee for supplying the 12D7 and
12D7-MDR1 cell lines and for critical advice and discussions. We thank
Drs J.A. Trapani, G.A. Pietersz, and S.M. Russell for helpful
discussions, and John Zalcberg and Phillip Kantharidis for reagents.
| |
FOOTNOTES |
Submitted July 17, 1998; accepted October 1, 1998.
R.W.J. is a CJ Martin Fellow of the National Health and Medical
Research Council of Australia. M.J.S. is currently supported by a
Wellcome Trust Australasian Senior Research Fellowship. This work was
supported by project grants from the National Health and Medical
Research Council of Australia.
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 Ricky W. Johnstone, PhD, The Austin
Research Institute, Austin Hospital, Studley Rd, Heidelberg 3084, Victoria, Australia; e-mail: r.johnstone{at}ari.unimelb.edu.au.
| |
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Top
Abstract
Introduction