From the Peter MacCallum Cancer Institute,
Trescowthick Research Laboratories, St. Andrews Place, East Melbourne
3002, Victoria, Australia, the ¶ Evanston Northwestern Healthcare
Research Institute, Evanston, Illinois 60201, and the
Rotary
Bone Marrow Research Laboratory, Royal Melbourne Hospital Research
Foundation, Parkville 3052, Victoria, Australia
Received for publication, November 29, 2000, and in revised form, February 15, 2001
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ABSTRACT |
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P-glycoprotein (P-gp) is an
ATP-dependent drug pump that confers multidrug resistance
(MDR). In addition to its ability to efflux toxins, P-gp can also
inhibit apoptosis induced by a wide array of cell death stimuli that
rely on activation of intracellular caspases for full function. We
therefore hypothesized that P-gp may have additional functions in
addition to its role in effluxing xenotoxins that could provide
protection to tumor cells against a host response. There have been a
number of contradictory reports concerning the role of P-gp in
regulating complement activation. Given the disparate results obtained
by different laboratories and our published results demonstrating that
P-gp does not affect cell death induced by another membranolytic
protein, perforin, we decided to assess the role of P-gp in regulating
cell lysis induced by a number of different pore-forming proteins.
Testing a variety of different P-gp-expressing MDR cell lines produced following exposure of cells to chemotherapeutic agents or by retroviral gene transduction in the complete absence of any drug selection, we
found no difference in sensitivity of P-gp+ve or
P-gp P-glycoprotein (P-gp),1
a member of the ATP-binding cassette (ABC) superfamily, is encoded by
the MDR1 gene in humans and mdr1a and
mdr1b in mice and has been demonstrated to act as a very
efficient toxin efflux molecule (1, 2). In the clinical setting,
expression of P-gp on tumor cells confers resistance to a wide range of
different chemotherapeutic agents constituting a multidrug-resistant
(MDR) phenotype and a poor prognosis. The current working model
maintains that P-gp removes xenotoxins in an energy
(ATP)-dependent manner by intercepting the drug as it moves
through the lipid membrane and flips the drug from the inner leaflet to
the outer leaflet and into the extracellular media (3). Consistent with
its toxin clearance role, P-gp is expressed on the surface of normal
human cells found in the gut, liver, and kidney tubules, and at
blood-tissue barriers (4). However, P-gp is also expressed in the
adrenal gland, hemopoietic stem cells, natural killer (NK) cells,
antigen-presenting dendritic cells, and T and B lymphocytes (5, 6), and
a role for P-gp in removing xenotoxins from these cells is not
immediately apparent.
Recent studies by our group and others have indicated that, in addition
to its role as an efflux pump, P-gp also regulates programmed cell
death mediated by some chemotherapeutic drugs, serum starvation,
ultraviolet (UV) irradiation, and ligation of the cell surface death
receptors Fas and tumor necrosis factor receptor (7-9). The ability of
P-gp to inhibit cell death mediated by these diverse apoptotic stimuli
appears to be due to P-gp-mediated inhibition of caspase activation. In
contrast, other stimuli such as the chemotherapeutic agent
hexamethylene bisacetamide (10), the protein kinase C inhibitor
staurosporine (11), the CTL granule protein granzyme B (9) and the CTL
pore-forming protein perforin (9), which are fully functional in the
absence of caspase activation, were not affected by P-gp.
A number of groups have analyzed the potential effects of P-gp on cell
lysis mediated by complement, a family of related pore-forming proteins
necessary for antibody-mediated cytolysis. Initial studies focused on
successfully using anti-P-gp antibodies and rabbit complement to purge
autologous bone marrow grafts of residual P-gp+ve MDR cells
(12), or complement fixing anti-P-gp antibodies to eliminate
P-gp+ve tumors in mouse model systems (13). In agreement
with these studies were those of Bomstein and Fishelson (14), who
demonstrated that P-gp+ve tumor cell lines were more
sensitive to complement-mediated lysis compared with matched
P-gp Perforin is essential for death of virus-infected or malignant target
cells by CTL and NK effector cells (18). Perforin, expressed in the
granules of CTL and NK cells mediates the transport of granzymes into
the target cell to induce programmed cell death via a process known as
granule exocytosis (18). Following the initial cloning and
characterization of perforin, it was demonstrated that there were
structural and functional similarities between perforin and the C9
component of complement (19, 20). Both could form pores in lipid
membranes, both had conserved amino acid motifs, and monoclonal
antibodies raised against purified C9 and perforin showed
cross-reactivity (19, 20). We have previously demonstrated equivalent
lysis of P-gp+ve and P-gp Cell Culture--
The acute T cell leukemia cell line, CEM-CCRF,
its doxorubicin (DOX)-selected and -resistant P-gp+ve line
CEM-A7+, have been previously described (21). K562 and
vincristine-selected P-gp+ve Kvin2000 cells were kindly
provided by Greg Woods (University of Tasmania, Hobart, Australia). All
cells were grown in RPMI medium 1640 supplemented with 10% (v/v) fetal
calf serum, 2 mM glutamine, 100 units/ml penicillin, and
100 µ g/ml streptomycin (Life Technologies, Inc.). The cell surface
expression of P-gp and Fas was confirmed and monitored by fluorescence
analysis using the MRK 16 anti-P-gp (Kamiya Biochemical Co., Thousand
Oaks, CA) and CH-11 anti-human Fas (Upstate Biotechnology Inc., Lake
Placid, NY) monoclonal antibodies, respectively. Drug efflux activities of P-gp and reversal with verapamil were assessed by rhodamine 123 exclusion assays as described (22).
Generation of MSCV-based Supernatant and Transduction of
Mammalian Cell Lines--
The MDR1 coding region was cloned into the
retroviral vector plasmid MSCV. This bicistronic vector contains (i)
the amphotropic retrovirus murine stem cell virus (MSCV) 5'-long
terminal repeat, (ii) the encephalomyocarditis internal ribosomal entry
site, (iii) the green fluorescent protein (GFP) cDNA, and (iv) the
MSCV 3'-long terminal repeat (23). The plasmid was cotransfected with
an amphotropic packaging plasmid into 293T cells by calcium phosphate precipitation. After 48 h, the supernatant containing amphotropic particles was harvested, filtered, and added to CEM-CCRF cells every
8 h for 3 days. The cells were allowed to recover for 72 h
and then analyzed for GFP expression by flow cytometry. The highest
expressing 10% of cells were sterilely sorted, expanded, resorted, and
subsequently expanded in oligoclonal pools. Cells were subsequently
screened for expression of P-gp and sorted by flow cytometry using the
MRK-16 monoclonal antibody (Kamiya Biochemical Co.).
Membramolytic and Chemotherapeutic Agents--
Human perforin
was purified as described previously (24). Pneumococcal PLO was
provided by Dr. James Paton (Childrens and Womens Hospital, Adelaide,
Australia). Freshly prepared serum from naïve rabbits was used
as the source of hemolytic complement. Small aliquots of serum were
stored at Expression of Cell Surface CD35, CD46, CD55, and
CD59--
Expression of the regulators of complement activation, CD35,
CD46, CD55. and CD59, on the cell surface was determined by flow cytometry. Monoclonal antibodies against CD35 (BD PharMingen, San
Diego, CA), CD46 (E4.3, IgG2a), CD55 (clone 1H4, IgG1), and CD59 (clone
MEM-43, IgG2a), kindly provided by Dr. S. Russell (Peter MacCallum
Cancer Institute, Melbourne, Australia), or appropriate isotype control
antibodies were incubated with P-gp+ve and
P-gp Cytotoxicity Assays--
Cell death was assessed by
51Cr release assays as described (8). The spontaneous
release of 51Cr was determined by incubating the target
cells with medium alone (or in the presence of anti-P-gp monoclonal
antibody (mAb), 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 SDS to a final
concentration of 5%. The percentage of specific lysis was calculated
as follows: 100 × [(experimental release
Complement-mediated lysis was assessed by treating 5 × 104
51Cr-labeled target cells with diluted active or
heat-inactivated rabbit complement for 10-60 min at 37 °C in the
presence or absence of anti-CD71 (clone M-A712, IgG2a, BD PharMingen)
or anti-Fc P-gp Does Not Protect K562 Cells against Complement-mediated
Lysis--
A previous study had shown that P-gp+ve K562
cells were less sensitive to complement-mediated lysis compared with
parental P-gp
Complement activation can be inhibited by cell surface regulators of
complement activation (RCA) proteins including CD35 (CR1), CD46 (MCP),
CD55 (decay accelerating factor), and CD59 (17). To determine whether
P-gp+ve and P-gp P-gp Does Not Affect Cell Lysis Induced by Other Pore-forming
Proteins--
To determine whether P-gp+ve and
P-gp P-gp Does Not Protect T Cell Lines against Membrane Lysis Induced
by Different Pore-forming Proteins--
To ensure that the results
obtained using the K562 erythoroblastoid lines were not specific to
myeloid cells, P-gp+ve (A7) and P-gp
The P-gp+ve K562 and CEM cells used in the studies
presented thus far were both produced following treatment of parental
cells with vincristine and doxorubicin, respectively. It is therefore possible that any affect that P-gp may have on cell lysis induced by
complement or other pore-forming proteins may be negated by other
genetic defects introduced into these cells following drug treatment.
To overcome this potential problem, we expressed P-gp on the surface of
CEM cells by retroviral gene transduction in the complete absence of
drug selection. CEM cells were transduced with virus expressing GFP and
P-gp and were sorted by flow cytometry. Thus, cell lines expressing
P-gp and/or GFP were obtained and used in the following studies.
Expression of cell surface P-gp and intracellular GFP in CEM, A7, and
retroviral transduced CEM cells was assessed by flow cytometry (Fig.
6A). P-gp was expressed on A7
and fluorescence-activated cell-sorted RP-gp+ve
(GFP+ve/P-gp+ve) and not on CEM or
RP-gp
To determine whether P-gp expressed on the retroviral transduced cells
confers MDR, the CEM, A7, RP-gp+ve, and
RP-gp
We next assessed the sensitivity of RP-gp+ve and
RP-gp The role of P-gp in regulating complement-mediated cell lysis has
been a controversial one with different groups reporting conflicting
data. One group correlated P-gp expression with increased sensitivity
of cells to complement-mediated cell lysis (14). In contrast, two
separate studies by another group demonstrated that functional P-gp
inhibited the rate of complement deposition on the cell surface and
provided evidence suggesting that P-gp inhibited the rate of formation
of the complement MAC (15, 16). In an attempt to clarify this issue, we
tested a number of different P-gp+ve and
P-gp Although the role of P-gp in effluxing xenotoxins out of cells has long
been established, the physiological functions of P-gp have yet to be
identified. Analysis of MDR1 gene knockout mice reveals an
inability to clear certain neurotoxins (26, 27), and the mice
spontaneously develop ulcerative colitis, possibly the result of an
inability to remove bacterial toxins produced by the resident mucosal
flora (28). Using in vitro systems, we and others have
demonstrated additional roles for P-gp including regulation of caspase
activation (8, 9), chloride channel activity (29-31), lipid transport
(32), and intracellular cholesterol trafficking (33, 34). Expression of
P-gp correlates with an increase in intracellular pH
(pHi) and decreased plasma membrane potential
(Vmax) (35). It has been proposed by Roepe and
colleagues (36) that this effect on cell physiology indirectly affects
the function of chemotherapeutic drugs that are not directly pumped by
P-gp by "partitioning" drugs away from their intracellular targets.
As stated above, a role for P-gp in positively or negatively regulating
complement-mediated cell lysis has also been proposed. Whether or not
any of these proposed roles of P-gp represent true physiological
functions of the molecule remains to be thoroughly tested.
It is presently unclear how or why different laboratories could produce
such divergent findings with regards to the role of P-gp in regulating
complement activation. The studies by Bomstein and Fichelson (14) may
merely correlate P-gp expression with increased sensitivity to
complement-induced lysis. The authors did block P-gp function by
addition of anti-P-gp mAb, resulting in a decrease in sensitivity of
cells to complement attack. However, the MRK-16 antibody used (IgG2a)
is capable of activating complement (13), and one might have expected
an increase in complement-mediated lysis or even a zero net effect
rather than the decrease in complement activity observed.
Interpretation of the results from these studies were made somewhat
difficult, given the differences in expression of certain complement
regulatory proteins observed between P-gp+ve and
P-gp The studies by Weisberg and colleagues (15, 16) showing P-gp-mediated
resistance to complement-induced cell lysis appeared well controlled,
although the relative expression of complement regulatory proteins such
as CD35, CD46, CD55, and CD59 on the different P-gp+ve and
P-gp A number of experiments performed in a variety of different
laboratories have provided evidence for addition roles for P-gp other
than its ability to efflux xenotoxins (see Ref. 37 and references
therein). The putative role of P-gp in regulating cell lysis mediated
by pore-forming proteins on the cell surface has been the topic of some
debate, and we have attempted to answer this question using a variety
of different cell lines expressing P-gp tested against a range of
different membranolytic proteins. From our data, we conclude that P-gp
does not regulate cell lysis induced by activated complement, purified
human perforin, or pneumolysin.
ve cells to the pore-forming proteins complement,
perforin, or pneumolysin. Based on these results, we conclude that P-gp
does not affect cell lysis induced by pore-forming proteins.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ve parental cells. However, in contrast to these
findings were two reports demonstrating a role for functional P-gp in
inhibiting membrane damage by complement (15, 16). The authors of these studies presented evidence suggesting that P-gp-mediated intracellular alkalinization (pHi) and/or decreased plasma
membrane potential (Vmax) resulted in a
reduction in the rate of formation of the "membrane attack complex"
(MAC) at the cell surface. Functional MACs consist of complement
components C5b, C6, C7, C8, and multiple C9 molecules to form
pores of ~100 Å (17). Expression of functional P-gp was shown
to correlate with a reduction in the rate of MAC formation rather than
the molecular stoichiometry of MAC complexes (16). It was hypothesized
that altered pHi and/or
Vmax may somehow directly or indirectly affect
C9 polymerization, resulting in a net loss of functional MAC formation
(16).
ve CEM cells treated
with perforin (9). In view of the structural and functional
similarities between C9 and perforin and the contradictory findings by
a number of groups regarding regulation of complement-mediated lysis by
P-gp, we performed a comprehensive analysis of the role of P-gp in
regulating pore formation. Using a number of matched P-gp
ve and P-gp+ve cell lines isolated
following treatment of cells with chemotherapeutic agents, and
different pore-forming proteins such as activated complement, purified
recombinant perforin, and the bacterial toxin pneumolysin, we observed
no effect of P-gp on cytolysis induced by these agents. These studies
were confirmed using P-gp+ve multidrug-resistant cells
produced by retroviral gene transduction and cloned by
fluorescence-activated cell sorting in the complete absence of drug
selection. Our data therefore indicate that P-gp does not affect the
cytolytic effects of pore-forming proteins.
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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70 °C and discarded after each use. Heat inactivation at
65 °C for 30 min caused total inhibition of the complement activity.
DOX and vincristine were obtained from Dr. Phillip Kantharidis (Peter
MacCallum Cancer Institute, East Melbourne, Australia).
ve CEM cells on ice for 1 h. Cells were washed
in phosphate-buffered saline containing 0.5% bovine serum albumin and
incubated with fluorescein isothiocyanate anti-mouse immunoglobulin for
1 h on ice. Cells were washed, and antibody binding was detected
by flow cytometry using a FACScan (Beckton Dickinson).
spontaneous
release)/(maximum release
spontaneous release)]. To inhibit
P-gp function, the labeled targets were preincubated for 30 min with
UIC2 (IgG2a mAb; final, 0.1-5 µg/ml) (Coulter, Miami, FL) or
verapamil (0.5-10 µM) (Knoll Australia, Lane Cove,
Australia) prior to the cytotoxicity assay. Isotype control antibodies
were included where applicable. To inhibit caspase activity, labeled
target cells were preincubated for an additional 30 min with peptidyl
fluoromethyl ketones (ZFA- and ZVAD-fluoromethyl ketones) (Enzyme
System Products, Dublin, CA) (final, 50 µM).
RII (clone 8.26, IgG2b) (25) monoclonal antibodies where
applicable. Lysis by perforin or pneumolysin was assessed by treating
5 × 104 51Cr-labeled target cells with diluted
pore-forming proteins for 60 min at 37 °C.
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ve K562 cells (15). In that study,
complement deposition was induced by activating the classical
complement pathway following addition of an anti-CD71 antibody (15). To
test this finding, we used matched P-gp
ve and
P-gp+ve K562 cells derived following incubation of the
parental cell line in vincristine, and anti-CD71 or anti-Fc
RII mAbs
to activate the complement cascade. Equivalent binding of both
antibodies to their target antigens on P-gp+ve and
P-gp
ve K562 cells was demonstrated (Fig.
1). Addition of a single dose of
complement to increasing concentrations of anti-CD71 (Fig. 2A, lanes 3-8) or
anti-Fc
RII (Fig. 2A, lanes 13-18) mAbs
resulted in a dose-dependent increase in cell lysis. Cell
death was dependent on activated complement, as heat inactivation of
the rabbit serum completely inhibited 51Cr release (Fig.
2A, lanes 9-12, 19, and
20). The drug efflux function of P-gp expressed on the K562
cells was confirmed by addition of DOX to P-gp+ve and
P-gp
ve cells. Only P-gp
ve K562 cells were
sensitive to DOX-induced cell death (Fig. 2A, lanes 21 and 22). To confirm these
data and demonstrate that the dose of complement used in Fig.
2A did not overwhelm potential subtle differences in
susceptibility of P-gp+ve and P-gp
ve K562
cells to complement-mediated lysis, a single dose of anti-Fc
RII antibody was added to increasing concentrations of complement (Fig.
2B). Both P-gp+ve and P-gp
ve K562
cells were equally susceptible to lysis over the entire complement
concentration range (Fig. 2B, lanes 5-12).
Once again, heat inactivation of the rabbit sera inhibited cell lysis
(Fig. 2B, lanes 13-16) and only the
P-gp
ve K562 cells were susceptible to drug-induced death
(Fig. 2B, lanes 17 and
18).
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Fig. 1.
Cell surface expression of CD71,
Fc RII, and P-gp on K562 and Kvin. Cells
were incubated with mAbs specific for CD71, Fc
RII and P-gp
(filled histograms) or appropriate isotype
controls (unfilled histograms), and fluorescein
isothiocyanate-labeled anti-mouse secondary antibodies. Cell surface
antibody binding was detected by flow cytometry.
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Fig. 2.
P-gp does not affect complement-mediated
lysis of myeloid cells. A, 51Cr-labeled
K562 (P-gp ve) and Kvin (P-gp+ve) cells were
incubated with rabbit serum containing active (C') or
heat-inactivated complement (HI C') in the presence or
absence of anti-CD71 or anti-Fc
RII monoclonal antibodies for 1 h at 37 °C. As a control for P-gp function, cells were treated with
doxorubicin (Dox, 100 ng/ml) for 24 h at 37 °C.
B, 51Cr-labeled K562 and Kvin cells were
incubated with anti-Fc
RII monoclonal antibody and serially diluted
(1:4-1:32) rabbit serum containing active complement (C')
or heat-treated rabbit serum (1:4) containing inactive complement
(HI C') for 1 h at 37 °C. As a control,
cells were treated with doxorubicin (Dox, 100 ng/ml) for
24 h at 37 °C. Data are calculated as the mean ± S.E. of
triplicate samples and are representative of a least two different
experiments.
ve K562 cells expressed
equivalent levels of RCA proteins, fluorescence analyses using mAbs
specific for the different RCA proteins were performed. As shown in
Fig. 3, no significant difference in
expression of CD35, CD46, CD55, or CD59 was observed between
P-gp+ve and P-gp
ve K562 cells.
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Fig. 3.
Cell surface expression of complement
regulatory proteins. K562 and Kvin cells were incubated with mAbs
specific for CD35, CD46, CD55, and CD59 (filled
histograms) or appropriate isotype controls
(unfilled histograms), and fluorescein
isothiocyanate-labeled anti-mouse secondary antibodies. Cell surface
antibody binding was detected by flow cytometry.
ve K562 cells were equally susceptible to death
induced by other pore-forming proteins, cells were treated with
purified perforin (Fig. 4A) or
pneumolysin (Fig. 4B). P-gp did not affect perforin- (Fig.
4A, lanes 1-12) or pneumolysin-mediated cell
lysis (Fig. 4B, lanes 1-10), but did protect
K562 cells against cell death induced by the chemotherapeutic agents
vincristine (Fig. 4A, lanes 13 and 14)
and doxorubicin (Fig. 4B, lanes 11 and
12). These experiments were performed numerous times, and no
statistically significant difference in susceptibility to death induced
by these agents was observed in P-gp+ve versus
P-gp
ve cells. These data therefore demonstrate that
functional P-gp does not protect myeloid cells against cell lysis
induced by a range of different membranolytic proteins.
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Fig. 4.
P-gp does not affect myeloid cell lysis
induced by perforin or pneumolysin. 51Cr-Labeled K562
(P-gp ve) and Kvin (P-gp+ve) cells were
incubated with purified human perforin (Pfp, 1.0 hemolytic
units/ml serially diluted 2-fold) for 4 h at 37 °C or
vincristine (Vin, 100 ng/ml) 24 h at 37 °C
(A) and PLO (1.44 µg/ml serially diluted 10-fold) for
4 h at 37 °C or doxorubicin (Dox, 100 ng/ml) for
24 h at 37 °C (B). Data are calculated as the
mean ± S.E. of triplicate samples and are representative of least
two different experiments.
ve (CEM)
T lymphoid cell lines were used. Cells were treated with activated
complement (Fig. 5A), purified
perforin (Fig. 5B), and pneumolysin (Fig. 5C) and
cell lysis assessed by 51Cr release assays. In all cases,
both P-gp+ve and P-gp
ve CEM cells were
equivalently susceptible to cell lysis induced by active pore-forming
proteins. In contrast, only P-gp
ve CEM cells were
susceptible to death induced by chemotherapeutic agents vincristine
(Fig. 5, A and B) or doxorubicin (Fig.
5C). It should be noted that the rabbit sera used in this
study contained significant levels of natural antibodies against both
P-gp+ve and P-gp
ve CEM cell lines as assessed
by flow cytometry (data not shown). This thereby eliminated the
necessity for anti-CD71 or anti-Fc
RII mAbs to be used in the
complement activation assays. In addition, there was no significant
difference in expression of cell surface CD35, CD46, CD55, and CD59
between CEM and A7 cells (data not shown).
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Fig. 5.
P-gp does not affect T lymphocyte cell lysis
induced by complement, perforin, or pneumolysin.
51Cr-Labeled CEM (P-gp ve) and A7
(P-gp+ve) cells were incubated with serially diluted
(A) rabbit serum containing active complement
(C', 1:4-1:64) or heat- inactivated rabbit serum (HI C', 1:4) for 1 h at
37 °C (A); purified human perforin (Pfp, 1.0 hemolytic units/ml serially diluted 2-fold) (B); or PLO
(1.44 µg/ml serially diluted 10-fold) for 4 h at 37 °C
(C). As a control for P-gp function, cells were treated with
vincristine (Vin, 100 ng/ml) (A) or doxorubicin
(Dox, 100 ng/ml) for 24 h at 37 °C (B and
C). Data are calculated as the mean ± S.E. of
triplicate samples and are representative of a least two different
experiments.
ve (GFP+ve/P-gp
ve) cells
(Fig. 6A). The efflux function of P-gp was determined by
123Rh efflux assays (Fig. 6B). As
123Rh, a demonstrated substrate for P-gp, is a fluorescent
dye, an increase in fluorescence following incubation with cells
indicates dye uptake (22). Following treatment of cells with
123Rh, A7 and RP-gp+ve showed only a marginal
increase in fluorescence over background compared with that seen with
CEM and RP-gp
ve cells (Fig. 6B). These data
indicate that P-gp expressed on the surface of RP-gp+ve
cells is capable of effluxing P-gp substrates.
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Fig. 6.
Production of P-gp-expressing CEM cells by
retroviral gene transduction. CEM cells were transduced with the
MSCV-MDR1 retrovirus, sorted for GFP expression by flow cytometry, and
then resorted for GFP expression ± expression of P-gp using the
MRK 16 anti-P-gp monoclonal antibody. Cells expressing GFP and P-gp
(RP-gp+ve) or GFP without P-gp (RP-gp ve) were
obtained. A, CEM, A7, RP-gp+ve, and
RP-gp
ve cells were analyzed for expression of cell
surface P-gp (vertical axis) or intracellular GFP
horizontal (horizontal axis). B, CEM,
A7, RP-gp+ve, and RP-gp
ve cells were assessed
for P-gp function by 123Rh efflux assays. Fluorescence of
resting (filled histograms) and
123Rh-treated (unfilled histograms)
cells was analyzed by flow cytometry. An increase in fluorescence
correlates with uptake of the fluorescent 123Rh dye.
ve cells were treated with chemotherapeutic agents
vincristine and doxorubicin (Fig. 7).
Both A7 and RP-gp+ve cells were resistant to various doses
of vincristine and doxorubicin, whereas CEM and RP-gp
ve
cells were sensitive to death induced by these agents. These data
indicate that RP-gp+ve cells express functional P-gp and
that P-gp expression alone is sufficient to induce an MDR phenotype in
the CEM T cell line.
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Fig. 7.
P-gp-expressing CEM cells produced by
retroviral gene transduction are multidrug-resistant.
51Cr-Labeled CEM, A7, RP-gp+ve, and
RP-gp ve cells were treated with various concentrations of
vincristine (Vin) or doxorubicin (Dox) for
24 h at 37 °C. Data are calculated as the mean ± S.E. of
triplicate samples and are representative of least two different
experiments.
ve cells to cell lysis mediated by pneumolysin (Fig.
8A) and complement (Fig. 8B). Both cell lines were equivalently sensitive to death
induced by these membranolytic agents, with RP-gp+ve cells
demonstrating resistance to death induced by vincristine as a control
for P-gp function in these assays (Fig. 8, A and B, lane 14). Both RP-gp+ve
and RP-gp
ve cells expressed approximately equal levels of
cell surface CD35, CD46, CD55, and CD59 (data not shown). To determine
whether RP-gp+ve and RP-gp
ve cells may be
lysed by activated complement with different kinetics, time-course
experiments were performed. As shown in Fig. 8C,
RP-gp+ve and RP-gp
ve cells were lysed by
activated complement with very similar kinetics, indicating that, in
this in vitro system, functional P-gp does not significantly
affect the rate of complement deposition.
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Fig. 8.
RP-gp+ve cells are sensitive to
lysis induced by pore-forming proteins. A and
B, 51Cr-Labeled RP-gp ve and
RP-gp+ve cells were incubated with PLO (1.44 µg/ml
serially diluted 10-fold) for 4 h at 37 °C (A) or
serially diluted rabbit serum containing active complement
(C', 1:4-1:64) or heat-inactivated rabbit serum (HI
C', 1:4) for 1 h at 37 °C (B). C, to
analyze the kinetics of complement-mediated cell lysis, 51Cr-labeled RP-gp
ve and
RP-gp+ve cells were incubated with active (C',
1:4 and 1:8) or inactive (HI C', 1:4) complement and
membrane damage was measured over a 2-h time course. As a control for
P-gp function, cells were treated with vincristine (Vin, 100 ng/ml) for 24 h at 37 °C. Data are calculated as the mean ± S.E. of triplicate samples and are representative of least two
different experiments.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ve cell lines, including cells transduced with
retrovirus and selected in the absence of drug treatment, for
sensitivity to cell membrane damage mediated by pore-forming proteins.
The retroviral transduced P-gp+ve cells expressed levels of
P-gp equivalent to that seen in drug selected CEM-A7 and Kvin cells and
were equally insensitive to various chemotherapeutic drugs. This
indicates that, in this system, expression of P-gp was necessary and
sufficient to induce a multidrug-resistant phenotype. Cell lysis was
induced with a diverse range of membranolytic agents including
activated complement, purified human perforin, and the bacterial
pore-forming protein pneumolysin. Our data indicate that functional
P-gp does not significantly affect the rate or degree of membrane lysis
irrespective of cell type, the manner by which P-gp expression was
achieved, or the lytic agent used.
ve cells.
ve cell lines was not assessed. We attempted to
reproduce these findings using the same cell type (K562) and complement
activating antibodies directed against the same cell surface antigen
(CD71) used in those studies. However, we observed no difference in
complement sensitivity between P-gp+ve and
P-gp
ve cells. The cell lines used in our study expressed
similar levels of complement regulatory proteins, and the drug efflux
function of P-gp was confirmed in every assay. Weisberg et
al. argue that altered intracellular pH may be the mechanism by
which P-gp can affect complement-induced cell death and hypothesize
that other pore-forming proteins such as a complement C9-like protein
produced by Trypanosoma cruzi may be similarly affected. No
data have yet been published supporting this hypothesis. In contrast,
we demonstrate in our assay systems that neither purified human
perforin which is also homologous to complement C9, nor the
pneumococcal pore-forming protein, pneumolysin, was affected by
functional P-gp. We have quantitated the pHi of
our cells using the carboxy SNARF-1 acetate fluorescent dye (Molecular
Probes) and flow cytometry. We observed an increase in intracellular pH
in P-gp+ve (CEM-A7 = pHi 7.5, Kvin = pHi 7.7) compared with
P-gp
ve cells (CCRF-CEM = pHi 7.2, K562 = pHi 7.5); however, no commensurate difference in
membrane disruption by pore-formers was observed.
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ACKNOWLEDGEMENTS |
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We thank Drs. J. A. Trapani, V. Sutton, and S. M. Russell for helpful discussions and Dr. J. Patton for valuable reagents.
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FOOTNOTES |
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* This work was supported in part by grants from the National Health and Medical Research Council of Australia, the Anti-Cancer Council of Victoria, the Wellcome Trust, the National Arthritis Foundation, and by National Institutes of Health Grant AI/GM 44941-01 IMB.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Wellcome Trust senior research fellow. To whom correspondence should be addressed. Tel.: 61-3-9656-3727; Fax: 61-3-9656-1411; E-mail: r.johnstone@pmci.unimelb.edu.au.
** Senior research fellow of the National Health and Medical Research Council of Australia.
Principal research fellow of the National Health and Medical
Research Council of Australia.
Published, JBC Papers in Press, February 20, 2001, DOI 10.1074/jbc.M010774200
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ABBREVIATIONS |
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The abbreviations used are: P-gp, P-glycoprotein; MDR, multidrug resistance; NK, natural killer; PLO, pneumolysin; MAC, membrane attack complex; CTL, cytotoxic T lymphocyte; DOX, doxorubicin; GFP, green fluorescence protein; mAb, monoclonal antibody; CR1, complement receptor 1; MCP, membrane cofactor protein; pHi, intracellular pH; Vmax, plasma membrane potential; MSCV, murine stem cell virus; RCA, regulators of complement activation.
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