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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 2 (January 15), 1999:
pp. 599-606
P-Glycoprotein Expression on Normal and Abnormally Expanded Natural
Killer Cells and Inhibition of P-Glycoprotein Function by Cyclosporin A
and Its Analogue, PSC833
By
Motoki Egashira,
Norihiko Kawamata,
Koichi Sugimoto,
Takako Kaneko, and
Kazuo Oshimi
From the Department of Hematology, Juntendo University School of
Medicine, Tokyo, Japan.
 |
ABSTRACT |
P-glycoprotein (P-gp), a transmembrane efflux pump encoded by the
MDR1 gene, has been found to be expressed in many normal bone
marrow and peripheral blood cells. Among normal leukocytes, CD3 CD16+ or
CD3CD56+ lymphocytes, ie, natural killer
(NK) cells, express relatively high levels of P-gp, but little is known
about P-gp in abnormally expanded NK cells. In this study, we examined
the expression and activity of P-gp on NK cells derived from three
normal donors, six patients with indolent NK cell-lineage granular
lymphocyte-proliferative disorder (NK-GLPD), three patients with
aggressive NK cell tumors (one NK cell leukemia and two nasal NK cell
lymphoma), and two NK cell lines. By flow cytometric analysis using the
monoclonal antibody (MoAb) MRK16 and rhodamine 123 dye (Rh123), P-gp
expression and the efflux of Rh123 were found in all NK samples except
one NK cell line. The Rh123 efflux of NK cells was inhibited by
cyclosporin A (CsA) and its analogue PSC 833, but the aggressive NK
tumor cells were less inhibited than were the other NK cells. The
percent inhibition of efflux in the normal NK cells, indolent NK-GLPD cells and aggressive NK cell tumors was 81.8% ± 0.9%, 93.4% ± 3.1% and 36.9% ± 11.7%, respectively, by 1 µmol/L CsA, and
80.2% ± 3.6%, 91.7% ± 2.6% and 32.7% ± 10.1%, respectively,
by 1 µmol/L PSC833. In reverse transcription-polymerase chain
reaction (RT-PCR) analysis, the low inhibitory effect of P-gp
modulators in aggressive NK cell tumors did not correlate to the
expression level of MDR1 gene, multidrug resistance-associated
protein gene, or human canalicular multispecific organic anion
transporter gene. This phenomenon could be related to the presence of
other transporters or to unknown cellular or membrane changes. Some
patients with NK cell tumors have been reported to show a highly
aggressive clinical course and to be refractory to chemotherapy, and
this could be related to the expression of P-gp on NK cells. Our
results suggest that, although the inhibitors for P-gp have been used
in combination with chemotherapy in some hematologic tumors, these
inhibitors may be less effective against aggressive NK cell tumors.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
IN TUMOR CELLS, the development of
resistance against multiple lipophilic cytotoxic drugs is a major
impediment to cancer chemotherapy. Such multidrug resistance (MDR) is
mediated by the multidrug transporter P-glycoprotein (P-gp), encoded by the MDR1 gene, which functions as an adenosine
triphosphate (ATP)-dependent drug efflux pump of broad
substrate specificity.1,2 P-gp is expressed not only in
neoplastic cells, but also in various normal cells including epithelial
cells of the gastrointestinal tract, adrenal gland, and biliary
canaliculi.3,4 Among normal blood cells,
CD34+ cells, CD56+ cells, and
CD8+ cells have been shown to display high levels of P-gp
expression.5-8 Natural killer (NK) cells usually express
CD56 antigen, so it is expected that NK cells express P-gp. To date,
however, P-gp expression on normal NK cell subsets has not
been reported, to our knowledge, and only a few studies have been
reported about the relationship between P-gp and NK cell
tumors.9-12
Since the discovery of the reversal effect of verapamil on
MDR,13 many agents that modulate P-gp function have been
identified, including several calcium antagonists, calmodulin
inhibitors, quinoline compounds, FK506, cyclosporin A (CsA), and its
analogue PSC833.14-17 CsA and PSC833, with chemotherapeutic
agents, have recently been used in the treatment of acute myeloid
leukemia (AML), non-Hodgkin's lymphoma, and multiple
myeloma.18-21 There are no reports describing the use of
these modulators in NK cell tumors to our knowledge.
In this study, to clarify whether the P-gp expression in NK cells is
carried into the malignancy originating from that cell type, we
analyzed the expression and function of P-gp on various NK cell
samples, including normal and abnormally expanded NK cells. We also
investigated the effect of the P-gp modulators CsA and PSC833 on these
NK cells.
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MATERIALS AND METHODS |
Isolation of NK cells.
From three healthy donors, peripheral blood mononuclear cells (PBMC)
were isolated from freshly drawn heparinized peripheral blood by
Ficoll-Conray density gradient centrifugation. To purify NK cells, the
PBMC were applied to columns of nylon-wool and incubated for 1 hour at
37°C. Nylon-wool nonadherent cells were eluted with warm medium and
added to Percoll (Pharmacia Fine Chemicals, Uppsala, Sweden) density
gradient solution mixed with 10% fetal calf serum (FCS) containing
RPMI 1640 medium (GIBCO BRL, Grand Island, NY). The Percoll solutions,
in volumes of 2 mL, were carefully layered into a 10-mL round-bottom
test tube, starting with 40% Percoll (fraction 5) and graded by 2.5%
concentration reductions to 30% Percoll on the top (fraction 1). After
the lymphocytes were added to the Percoll solution, they were
centrifuged at 650g for 20 minutes at room temperature. The
lymphocytes from low-density fractions 1 and 2 were mixed and used as
NK-rich lymphocytes. For the further purification of the NK cells,
CD3+ and CD20+ cells were removed from NK-rich
fractions by an immunomagnetic isolation technique. Patient NK cells
were purified from PBMC and lymph nodes by an immunomagnetic isolation
technique.
NK cell lines.
The NK cell line, NK92,22 was kindly provided by Dr
Jiang-Hong Gong (University of British Columbia, Vancouver, Canada). The NK cell line, NKL,23 was kindly provided by Dr Michael
J. Robertson (Harvard Medical School, Boston, MA). Both cell lines were
grown in RPMI 1640 supplemented with 15% FCS, 500 U/mL of interleukin-2 (IL-2; Shionogi, Tokyo, Japan), 100 U/mL penicillin and
100 µg/mL streptomycin.
Flow cytometric analysis of P-gp and NK-related antigens.
The expression of P-gp was measured by immunofluorescence using a
P-gp-reactive monoclonal antibody (MoAb), MRK16 (Kyowa Medex, Tokyo,
Japan). NK cells were stained with a three-color immunofluorescence method as follows. First, 5 × 105 cells were reacted
with 5 µg of MRK16 or IgG2a control antibody for 30 minutes at
4°C. After two washes with phosphate-buffered saline (PBS), the
cells were incubated with fluorescein isothiocyanate (FITC)-conjugated
F(ab')2 goat antimouse immunoglobulin (Ig). The cells were
washed twice and blocked with mouse IgG, then stained with
phycoerythrin-cyanine 5 (PC5)-conjugated anti-CD16 MoAb (Immunotech, Marseilles, France) and phycoerythrin (PE)-conjugated anti-CD56 MoAb
(Leu-19; Becton Dickinson, San Jose, CA). The P-gp expression on NK
cells was analyzed by flow cytometry (CYTORON ABOLUTE; Ortho Diagnostic
System, Raritan, NJ), gating on CD16+ or
CD56+ populations.
Measurement of Rh123 efflux.
The P-gp-mediated efflux in the NK cells was determined indirectly by
measuring the retention of a fluorescent P-gp substrate, rhodamine 123 dye (Rh123; Sigma, St Louis, MO), as previously described.7
Briefly, 5 × 105 cells were stained with 150 ng/mL
Rh123 for 15 minutes at 37°C and, after two washes, they were
incubated in 10 mL dye-free RPMI 1640 containing 10% FCS for 3 hours
at 37°C with or without P-gp inhibitors, 1 µmol/L CsA (Sandoz
Pharmaceuticals, Basel, Switzerland) or 1 µmol/L PSC833. PSC833 was a
kind gift from Sandoz. After the 3-hour efflux period, the cells were
labeled with PC5-conjugated anti-CD16 MoAb and PE-conjugated anti-CD56
MoAb, then washed, and immediately analyzed for fluorescence using the
flow cytometer gating on CD16+ or CD56+
populations. The mean channel fluorescence was measured for each sample
at the beginning (T0 hour) and end (T3 hour) of the assay. The percent
inhibition of efflux was calculated by incorporating the Rh123
fluorescence values into the following formula:
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Analysis of mRNA expression and quantification of polymerase chain
reaction (PCR) products.
The expression of MDR1 gene, multidrug resistance-associated
protein (MRP), human canalicular multispecific organic anion transporter (cMOAT), and  2-microglobulin (2m) were detected by
the reverse transcription (RT)-PCR method as previously
described.24-26 Total cellular RNA was extracted from the
purified NK cells by using the guanidium thiocyanate
method26 with Trizol (GIBCO BRL) according to the
manufacturer's protocol. cDNA was synthesized with 5 µg of total
cellular RNA using Ready To Go You-Prime First Strand Beads (Amersham
Pharmacia Biotech, Buckinghamshire, UK). cDNA derived from 50 ng of RNA
was next subjected to PCR for 35 cycles in a final volume of 100 µL
using 2.5 U Taq polymerase (Takara, Tokyo, Japan). The contents of
reaction mixtures have been described previously.26 The PCR
conditions are as follows: after an initial denaturation for 2 minutes
at 94°C, each cycle consisted of 30 seconds at 94°C, 30 seconds
at 55°C, and 60 seconds at 72°C. The sequences of the primers
were: MDR1 forward 5 -CCCATCATTGCAATAGCAGG-3 and
reverse 5-GTTCAAACTTCTGCTCCTGA-3; MRP forward
5-TGGGACTGGAATGTCACG-3 and reverse
5-AGGAATATGCCCCGACTTC-3; cMOAT forward
5-CTAATCTAGCCTACTCCTGC-3 and reverse
5-CTGCAGCTCTCTCTTCATGTGC-3; and 2m forward
5-ACCCCCACTGAAAAAGATGA-3 and reverse
5-ATCTTCAAACCTCCATGATG-3. The PCR products were 167, 293, 275, and 120 bp long, corresponding to MDR1, MRP, cMOAT and
2m, respectively. PCR products were separated on a 2% agarose gel.
The bands were visualized by ethidium bromide and photographed. For
quantification of MDR1 gene, the density of the bands was analyzed using BIO-CAPT V97 (Vilber Lourmat, Marne La Vallee, France)
and ZERO-Dscan (Scanalytics, Billerica, MA). Intensity of MDR1
expression was depicted as a ratio of MDR1 and 2m. The cell lines, K562/ADM and HepG2, were used for positive controls, K562/ADM for MDR1 and 2m, and HepG2 for MRP and cMOAT. These cell lines were kindly provided by Dr Toshiko Motoji (Department of
Hematology, Tokyo Women's Medical College, Tokyo, Japan).
| |
RESULTS |
Characterization of the patients studied.
The clinical findings of nine patients in our study are summarized in
Table 1. Patients 1 through 6 were
diagnosed to have indolent NK cell-lineage granular
lymphocyte-proliferative disorder (NK-GLPD), patient 7 had aggressive NK cell leukemia, and patients 8 and 9 had nasal NK cell
lymphoma. Patients 1 through 6 and 8 are described
elsewhere.27,28 Patient 9 showed leukemic change. The
classification into indolent and aggressive disorders was principally
based on their clinical course,27 but the phenotype of NK
cells, age, the presence of fever, and hepatosplenomegaly or
lymphadenopathy were somewhat helpful for the
classification.27,29 NK cells were isolated from PBMC in
patients 1 through 7 and 9 and from a metastatic lymph node in patient
8. None of the patients had received any treatment at the time when
their PBMC or lymph node cells were isolated. Their surface phenotypes
of PBMC or lymph node cells were CD16+ CD56+ in
patients 1 through 4 and 8, CD16+ CD56 in
patients 5 and 6, and CD16CD56+ in patients
7 and 9. All four patients tested with an indolent clinical course, and
only one of the three patients with aggressive clinical course
had high NK cell-mediated cytotoxicity against K562 target cells.
Patients 1 through 6 have been followed for at least 2 years and have
exhibited a stable clinical course without any treatment. Patients 7 through 9 had a progressive clinical course and died within 6 months
despite the administration of combination chemotherapy.
Expression and function of P-gp.
P-gp was expressed on all of the specimens tested except for one
NK-cell line, NKL (Fig 1). Two populations,
P-gp-positive and -negative, were present in NK cells in patient 6 (Fig 1i). In normal blood NK cells, it is said that three subsets are
present, CD16bright/CD56dim,
CD16dim/CD56bright, and
CD16neg/CD56bright.30 Because the
subset of CD16dim/CD56bright was a small
population, P-gp was measured in the following two subsets,
CD16+CD56+ and
CD16CD56+. In all three normal donors, no
difference was found in P-gp expression between these two subsets (Fig
1a through c).
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| Fig 1.
P-gp expression on NK cells. (a through c) are NK cells
from normal donors, (d through l) are those from patients, and (m) and
(n) are NK cell lines. (d through l) correspond to patients 1 through 9 of Table 1, respectively. (m) is the cell line NK92, and (n) is the
cell line NKL. Dark zone shows the results with MRK16 MoAb, and clear
zone shows the results with control IgG2a MoAb. In normal NK cells (a
through c), P-gp was measured in two subsets,
CD16+CD56+ (dark zone) and
CD16CD56+ (gray zone).
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P-gp function was measured by Rh123 efflux
(Fig 2). After the 3-hour efflux period,
P-gp-positive NK cells of all normal donors, indolent NK-GLPD, and
aggressive NK cell tumors and those of the NK cell line extruded Rh123,
but the P-gp-negative cell line, NKL, did not. Patient 6 had two
populations of NK cells, with the presence or absence of efflux
function (Fig 2i). In the three normal donors, no difference was found
in Rh123 efflux between the subsets of
CD16+CD56+ and
CD16CD56+ (data not shown).
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| Fig 2.
Two-dimensional flow cytometric dot plots of a
three-color flow cytometric assay of NK cells stained with CD16 and
CD56 after Rh123 efflux, gating on CD16+ or
CD56+ populations. (A, B, C, and D) are NK cells from the
normal donors, indolent NK-GLPD, aggressive NK cell tumors, and NK cell
lines, respectively. (d through l) correspond to patients 1 through 9 of Table 1, respectively. (m) is the cell line NK92, and (n) is the
cell line NKL. Dot density maps stained with Rh123 (T0), after the
3-hour efflux period without inhibitors (T3), and with 1 µmol/L
PSC833 (PSC833). The results with 1 µmol/L CsA are similar to those
with 1 µmol/L PSC833 and are omitted.
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Inhibition of P-gp function by CsA and PSC833.
Rh123 efflux was examined in the presence of the P-gp inhibitors, CsA
and PSC833 (Fig 2 and Table 2). When 1 µmol/L CsA or PSC833 was added, the percent inhibition of efflux in
the normal donors, indolent NK-GLPD, aggressive NK cell tumors, and the
cell line, NK92, were 81.8% ± 0.9%, 93.4% ± 3.1%, 36.9% ± 11.7% and 93.7%, respectively, by CsA and 80.2% ± 3.6%,
91.7% ± 2.6%, 32.7% ± 10.1% and 84.7%, respectively, by
PSC833, indicating that the P-gp of aggressive NK cell tumors was less
inhibited by CsA and PSC833 than were other NK cells.
Quantification of MDR1 gene.
Next, quantitative RT-PCR for MDR1 gene was performed to
examine whether the different effect of CsA and PSC833 on aggressive NK
cell tumors was due to a higher MDR1 gene expression.
MDR1 mRNA was detected on all the specimens tested except for
one NK-cell line, NKL (Fig 3), but the
aggressive NK cell tumors did not show higher MDR1 expression
than other NK cells (Fig 4).
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| Fig 3.
Analysis of mRNA expression of MDR1, MRP, cMOAT
and 2m genes using RT-PCR. Lane (N) is negative control, without
cDNA, and lane (P) is positive controls, K562/ADM for MDR1 and
2m, HepG2 for MRP and cMOAT. Lanes 1 through 3, 4 through 9, 10 through 12 and 13 through 14 are NK cells from the normal donors,
indolent NK-GLPD, aggressive NK cell tumors, and NK cell lines,
respectively. Lanes 4 through 12 correspond to patients 1 through 9 of Table 1, respectively. Lane 13 is the cell line NK92, and
lane 14 is the cell line NKL.
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| Fig 4.
Quantitative RT-PCR analysis for MDR1 gene.
Intensity of MDR1 expression is shown as a ratio of the density
of the bands for MDR1 and 2m. Lanes 1 through 3, 4 through
9, 10 through 12, and 13 and 14 are NK cells from the normal donors,
indolent NK-GLPD, aggressive NK cell tumors, and NK cell lines,
respectively. Lanes 4 through 12 correspond to patients 1 through 9 of
Table 1, respectively. Lane 13 is the cell line NK92, and lane 14 is
the cell line NKL.
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MRP and cMOAT expression.
To determine the presence of other non-P-gp transport mechanisms, MRP
and cMOAT were analyzed by RT-PCR (Fig 3). MRP was detected in NK cells
in two of the three normal donors, two of the six indolent NK-GLPD, one
of the three aggressive NK cell tumors, and both NK cell lines. cMOAT
was not detected in all of the NK cell samples tested. Thus, the low
inhibitory effect of P-gp modulators did not correlate with the
expression of MRP or cMOAT.
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DISCUSSION |
In this study, we examined the 14 NK cell samples (three normal, nine
abnormal, two cell lines), and all except one (the cell line, NKL)
expressed P-gp. In normal blood cells, as we and other investigators
have pointed out,6-8 CD56+ cells highly express
P-gp, but P-gp expression on NK cell subsets or their precursors has
not been reported.
Using human colon carcinoma and neuroblastoma cell lines, Mickley et
al31 and Bates et al32 found that the
expression of MDR1 gene was upregulated with differentiation.
NK cells are generated from CD34+ hematopoietic stem cells
under appropriate conditions in vitro and in vivo.33
Chaudhary et al5 have reported that CD34+ cells
express P-gp, but MDR1 expression in normal NK developmental pathway is still an enigma. In normal adult NK cells, it is said that
three subsets are present in the peripheral blood, ie,
CD16bright/CD56dim,
CD16dim/CD56bright, and
CD16neg/CD56bright, and NK cells seem to
differentiate inversely from the last to the first.30 We
investigated the differences of P-gp expression and Rh123 efflux among
the two subsets CD16+CD56+ and
CD16CD56+. In all three normal donors
tested, no difference was found between these two subsets (Fig 1 and
data not shown). In this view, it thus seemed that there was no
relationship between the differentiation and the P-gp expression in the
late stage of NK cell development.
Many patients with NK-GLPD have a stable clinical course, while some
have an aggressive clinical course.27,29 Among six patients
with indolent NK-GLPD, the phenotype of the NK cells in patients 1 through 4 was CD16+CD56+. These NK cells were
considered as mature NK cells and exhibited the same characteristics
with normal mature NK cells in terms of P-gp expression and function.
The phenotype of the NK cells in patients 5 and 6 was CD16+
CD56, and this phenotype is unusual in NK cells. Two
populations, P-gp-positive and P-gp-negative, existed in NK cells in
patient 6. P-gp-negative NK cells may exist in some particular
population of NK cells.
According to their clinical course, three patients were classified as
having aggressive NK cell tumors. As described before,28 we
considered the NK cells of case 8 as activated mature NK cells rather
than immature NK cells: these NK cells expressed activation markers
such as HLA-DR, CD71, CD45RO, CD30 and IL-2R , and strong NK
activity. On the other hand, NK cells of patients 7 and 9 did not
express CD16 antigen, nor did NK activity, suggesting that they were
derived from immature NK cells. The MDR1 gene expression level
of them was not particularly higher or lower than that of other NK
cells. However, because the sample size was too small, it will be
difficult to discuss the relationship between NK cell differentiation
and P-gp expression based on the present data.
Although the NK cell samples showed various degrees of P-gp expression,
all P-gp-positive NK cells functionally extruded Rh123. The
discordance between P-gp expression and functional Rh123 efflux, with
the failure of Rh123 efflux despite P-gp expression, has been reported
in some AML cases,34 but the P-gp-positive NK cells
observed in our study and others seemed to be always functional. Several investigators have described the relationship between P-gp
expression and NK cell-mediated cytolysis.35-38 Klimecki et al38 suggested that P-gp in NK cells functions as a
protective mechanism to remove perforin monomers from the
NK cell plasma membrane, or that P-gp is a transporter of cytotoxic
factors, which is involved in the killing process. In fact, evidence
exists that P-gp participates in the transport of cytokines (IL-2,
IL-4, and interferon- ) in normal peripheral T
lymphocytes.39 Our finding of P-gp expression and absent
cytotoxicity in patients 7 and 9, however, does not support previous
findings of a strong association between P-gp and NK cell function.
In preliminary experiments, the concentrations of CsA and PSC833 in the
culture were changed, and 1 µmol/L of CsA and PSC833 was found to
efficiently inhibit P-gp function in the cell line, NK92, and in NK
cells from one of the indolent NK-GLPD patients (data not shown). We
therefore used 1 µmol/L CsA and PSC833 in the present study. The P-gp
function in normal NK cells, indolent NK-GLPD cells, and the cell line,
NK92, was efficiently inhibited by 1 µmol/L CsA and PSC833, but
interestingly, P-gp function in the aggressive NK cell tumors could not
be inhibited as in the other NK cells (Fig 2 and Table 2). After
increasing the concentration of CsA and PSC833, we examined the Rh123
efflux in two of the three cases of aggressive NK cell tumors, patients
8 and 9. When 10 µmol/L CsA or PSC833 was added, the percent
inhibition of efflux was increased from 33% to 50% by CsA and from
22% to 65% by PSC833 in patient 8 and was increased from 50% to 65%
by CsA and from 42% to 67% by PSC833 in patient 9 (data not shown).
Thus, the aggressive NK cell tumors could not be inhibited sufficiently by even 10 µmol/L CsA and PSC833 and seem to be resistant to CsA and
PSC833. The cell line, NK92, was established from a patient with an
aggressive NK cell lymphoma, and the P-gp function of this cell line
was, in contrast, efficiently inhibited by 1 µmol/l CsA and PSC833.
It will be intriguing to examine the P-gp function of original NK cells
from this patient.
Several explanatory possibilities will be pointed out regarding the
question of why the P-gp function in the aggressive NK-cell tumors was
not sufficiently inhibited by CsA or PSC833. First, high MDR1
gene expression in aggressive NK cell tumors was expected. However,
aggressive NK cell tumors did not show higher MDR1 expression than other NK cells (Fig 4). Second, a mutant P-gp must be considered. Chen et al40 described a P-gp-positive human sarcoma cell
line, which was not modulated by CsA or PSC833. This cell line had a mutant MDR1 gene, which results in the deletion of the amino
acid phenylalanine at position 335 of P-gp (Phe335), and
they suggested that Phe335 was an important binding site on
P-gp for CsA and PSC833. We therefore investigated the sequence of DNA
from base pairs 1404-1501 containing codon 335 by PCR in patients 8 and
9, but a deletion of Phe335 was not found (data not shown).
Last, MDR mechanisms other than P-gp, such as MRP41 and
cMOAT,42 may participate in Rh123 efflux. MRP and cMOAT
also belong to the ATP binding cassette of drug transporter
proteins.41,42 MRP was detected in one of the three
aggressive NK cell tumors and also in several other NK cells. cMOAT was
not detected in all of the NK cell samples tested. Our Rh123 assay
results therefore seem to be unrelated to MRP and cMOAT, but the
involvement of other transporter proteins, such as MRP homologues
(MRP3, MRP4, and MRP5)43 and lung resistance-related protein,44 cannot be ruled out.
In summary, almost all NK cells expressed P-gp, and their P-gp was
functional. Some patients with NK cell tumors have been reported to
show a highly aggressive clinical course and to be refractory to
chemotherapy, and this could be related to the expression of P-gp in NK
cells. When chemotherapy is required for NK cell tumors, it may be
necessary to consider using anticancer drugs that are not a substrate
for P-gp. For the purpose of P-gp modulation, CsA and PSC833 have been
used in combination with chemotherapy in acute leukemia, multiple
myeloma, and malignant lymphoma,18-21 and this is an
alternative treatment for NK cell tumors, but our results suggest that
these inhibitors may be less effective in aggressive NK cell tumors due
to the presence of other transporters, or to unknown cellular or
membrane changes. Further studies are required to elucidate the
relationship between P-gp and NK cells.
| |
ACKNOWLEDGMENT |
We thank Dr Jiang-Hong Gong (University of British Columbia, Vancouver,
Canada), Dr Michael J. Robertson (Harvard Medical School, Boston, MA),
and Dr Toshiko Motoji (Department of Hematology, Tokyo Women's Medical
College, Tokyo, Japan) for cell lines. We thank Dr Katsuhiko Kitsugi
(Ortho Clinical Diagnostics, Tokyo, Japan) for technical advice.
| |
FOOTNOTES |
Submitted May 4, 1998;
accepted September 15, 1998.
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 Motoki Egashira, MD, Department of
Hematology, Juntendo University School of Medicine, 2-1-1 Hongo,
Bunkyo-ku, Tokyo 113-8421, Japan.
| |
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Abstract
Introduction