Uptake of cGMP into inside-out membrane vesicles prepared by a spontaneous, one-step vesiculation process is shown to be by a low affinity system that accounts for more than 80% of the tra
Trang 1cGMP and glutathione-conjugate transport in human erythrocytes
The roles of the multidrug resistance-associated proteins, MRP1, MRP4
and MRP5
Antonios Klokouzas†, Chung-Pu Wu, Hendrik W van Veen, Margery A Barrand and Stephen B Hladky
Department of Pharmacology, University of Cambridge, UK
The nature of cGMP transport in human erythrocytes, its
relationship to glutathione conjugate transport, and
pos-sible mediation by multidrug resistance-associated proteins
(MRPs) have been investigated MRP1, MRP4 and MRP5
are detected in immunoblotting studies with erythrocytes
MRP1and MRP5 are also detected in multidrug resistant
COR-L23/R and MOR/R cells but at greatly reduced levels
in the parent, drug sensitive COR-L23/P cells MRP4 is
detected in MOR/R but not COR-L23/R cells Uptake of
cGMP into inside-out membrane vesicles prepared by a
spontaneous, one-step vesiculation process is shown to be by
a low affinity system that accounts for more than 80% of the
transport at all concentrations above 3 lM This transport
is reduced by MRP inhibitors and substrates including
MK-571, methotrexate, estradiol 17-b-D-glucuronide, and S(2,4-dinitrophenyl)glutathione (DNP-SG) and also by glibenclamide and frusemide but not by the monoclonal Ig QCRL-3 that inhibits high-affinity transport of DNP-SG by MRP1 It is concluded that the cGMP exporter is distinct from MRP1and has properties similar to those reported for MRP4 Furthermore the evidence suggests that the protein responsible for cGMP transport is the same as that mediating low-affinity DNP-SG transport in human erythrocytes
Keywords: multidrug resistance-associated protein; guano-sine cyclic mono-phosphate transport; glutathione-conju-gate transport; human erythrocytes; membrane vesicles
Active transport of the cyclic nucleotide cGMP across
human erythrocyte membranes can be demonstrated using
intact cells [1] or inside-out membrane vesicle preparations
[2–4] In the studies using inside-out membrane vesicles, the
active uptake of cGMP was found to be saturable with two
components, one of high-affinity (Km 2–5 lM) [2,5] and
another of low-affinity (Km170 ± 50 lM) [5] Two
com-ponents have also been described for the transport of
another organic anion, a glutathione conjugate
S(2,4-dinitrophenyl)glutathione (DNP-SG), in human
erythro-cytes [6,7]
Two members of the multidrug resistance-associated protein (MRP) transporter family, MRP1and MRP5 have been detected previously in human erythrocyte membranes [8,9], and transport by MRP1has been conclusively shown
to account for the high-affinity component of DNP-SG transport [10–12] MRP4 and MRP5 have been shown to transport the cyclic nucleotides, cAMP and cGMP [9,13,14] and it has been suggested that MRP5 mediates the high-affinity component of the cGMP transport [15] However, the same group has questioned this identification [16] and recently it has been shown that when expressed in HEK293 cells, MRP4 and MRP5 mediate low-affinity transport of cyclic nucleotides [17]
The aim of the present study was to investigate the nature
of cGMP transport in human erythrocytes, its relationship
to glutathione conjugate transport, particularly to the low-affinity DNP-SG component, and its possible mediation by MRP4 and/or MRP5 The present work provides evidence from immunoblotting studies that both MRP5 [9] and MRP4 are expressed in human erythrocytes Using inside-out membrane vesicles prepared by a spontaneous, one-step vesiculation process, we identify a low affinity component for the cGMP transport which accounts for more than 80%
of the transport at all concentrations above 3 lM This transport is reduced by a range of inhibitors and substrates for MRPs including MK-571, methotrexate, E217bG, and DNP-SG and also by glibenclamide and frusemide We show that this cGMP exporter is distinct from MRP1and has characteristics similar to those reported for MRP4 The evidence suggests that the protein responsible for cGMP transport is the same as that mediating low-affinity DNP-SG transport in human erythrocytes
Correspondence to S B Hladky, Department of Pharmacology,
University of Cambridge, Cambridge, CB2 1PD, UK.
Fax: + 44 1223 334040, Tel.: + 44 1223 334019,
E-mail: sbh1@cam.ac.uk
Abbreviations: ATP-c-S, adenosine 5¢-O-(3-thiotriphosphate);
DNP-SG, S(2,4-dinitrophenyl)glutathione; E217bG, estradiol
17-b-D -glucuronide; GSH, reduced glutathione; HRP, horseradish
peroxi-dase; IBMX, isobutylmethylxanthine; MK-571,
(3-([[3-(2-[7-chloro-
2-quinolinyl]ethenyl)phenyl}-{(3-(dimethylamino-3-oxopropyl)-thio}-methyl]thio) propanoic acid; MRP, multidrug
resistance-asso-ciated protein; PNGase F, peptide N-glycosidase F; Ro, 31–8220
bisindolylmaleimide; SITS,
4-acetamido-4¢-isothiocyano-2,2¢-disulf-onic stilbene IX methanesulfonate.
Present address: Laboratory of Cell Biology, National Cancer
Institute, Building 37, Room 1B17, 37 Convent Drive, Bethesda,
MD 20892, USA.
Note: web page available at http://www.phar.cam.ac.uk
(Received 20 May 2003, revised 14 July 2003, accepted 15 July 2003)
Trang 2Experimental procedures
Chemicals
[8-3H]cGMP (specific activity 13.9 CiÆmmol)1) was obtained
from Amersham Biosciences, [glycine-2-3H]GSH (specific
activity 40 CiÆmmol)1) and [3H]glibenclamide (specific
acti-vity 44.7 CiÆmmol)1) were obtained from New England
Nuclear, respectively
M5I-1mAb against MRP5 was a kind gift of R J
Scheper (Free University, Amsterdam, the Netherlands);
anti-MRP4 mAb was a kind gift of G D Kruh (Fox Chase
Cancer Centre, Philadelphia, PA, USA); QCRL-3 mAb was
purchased from Signet Laboratories, USA M5I-1and
anti-MRP4 mAbs have been previously described [18,19]
4-Acetamido-4¢-isothiocyano-2, 2¢-disulfonic stilbene
(SITS), adenosine 3¢,5¢-cyclic monophosphate (cAMP),
adenosine 5¢-O-(3-thiotriphosphate) (ATP-c-S), adenosine
triphosphate (ATP), 4-aminopyridine, aprotinin,
1-chloro-2,4-dinitrobenzene, clotrimazole, creatine kinase, creatine
phosphokinase, daunorubicin, dideoxyforskolin,
iso-butylmethylxanthine (IBMX), doxorubicin, estradiol
17-b-D-glucuronide, forskolin, glibenclamide, glutathione
(reduced form, GSH), glutathione S-transferase, guanosine
3¢,5¢-cyclic monophosphate (cGMP), imidazole,
indometh-acin, leupeptin, lithocholic acid 3-sulphate, methotrexate,
pepstatin A, probenecid, taurocholic acid,
tetraethylammo-nium chloride, Triton X-100, Tween 20, verapamil and
vincristine were all obtained from Sigma Chemicals Calcein
was purchased from Molecular Probes Staurosporine and
bisindolylmaleimide IX methanesulfonate (Ro 31–8220)
were obtained from Calbiochem
(3-([[3-(2-[7-chloro-2-qui-
nolinyl]ethenyl)phenyl}-{(3-(dimethylamino-3-oxopro-pyl)-thio}-methyl]thio) propanoic acid, MK-571, was a
generous gift of M Turner (Merck-Frosst Center for
Therapeutic Research, Quebec, Canada) Peptide
N-glyco-sidase F (PNGase F) was purchased from Promega
Drugs were prepared in 10 mM Tris/HCl (pH 7.4) for
GSH, ATP, ATP-c-S, cGMP, taurocholic acid, imidazole,
vincristine and MK-571or in 66% dimethyl sulfoxide/34%
water for glibenclamide, SITS, methotrexate, verapamil,
indomethacin, E217bG and clotrimazole The final
concen-tration of the dimethyl sulfoxide did not exceed 0.5% in
each experiment GSH stock solutions (adjusted to pH 7.4)
were freshly prepared on the day of each experiment
[3H]DNP-SG was synthesized enzymatically as
previ-ously described [12,20] The purity of the3H-labelled
DNP-SG was determined by thin-layer chromatography on silica
gel plates [(0.25· 40 · 80) mm, AlugramSIL G/UV254,
Macherey-Nagel, Germany] using n-propanol:water (7 : 3,
v/v) as solvent [21]
Cell lines
COR-L23/R and MOR/R are MRP1-overexpressing,
multi-drug-resistant, human large-cell lung tumour lines produced
by doxorubicin selection [22,23] All cells were cultured on
plastic in growth medium containing RPMI-1640 medium
supplemented with 10% (v/v) foetal bovine serum, glutamine
(2 mM), penicillin (100 IUÆmL)1) and streptomycin
(100 lgÆmL)1) (complete RPMI-1640) in a 5% CO2
humi-dified incubator at 37C The L23/R and MOR/R sublines
were maintained in the presence of 0.2 lgÆmL)1 and 0.4 lgÆmL)1doxorubicin, respectively The cells were kept
in drug-free medium for at least 48 h before use in experiments Cells were passaged when they became conflu-ent RNA protection assay of the doxorubicin-resistant COR-L23/R and MOR/R cells [24] shows that these cells: do not express P-glycoprotein; over-express MRP1when com-pared to the doxorubicin-sensitive controls; and express MRP4 at a low level MRP5 is expressed at very low level in COR-L23 cells but at high level in MOR cells
Preparation of inside-out human lung tumour cell membrane vesicles
Membrane vesicles from human lung tumour cells were prepared according to a method described previously [25]
in the presence of protease inhibitors (5 lgÆmL)1leupeptin,
2 lgÆmL)1aprotinin, 80 ngÆmL)1pepstatin A) Briefly, cells were lysed in ice-cold hypotonic buffer (1mM Tris/HCl,
pH 7.4) for 30 min at 4C Following centrifugation
at 100 000 g for 30 min at 4C, the resulting pellet was homogenized vigorously with a Teflon hand homogenizer in buffer containing 10 mM Tris/HCl, 250 mM sucrose, and protease inhibitors, layered over 38% (w/v) sucrose in
1 0 mMTris/HCl and centrifuged at 100 000 g for 30 min at
4C The membranous material in the layer at the interface with the sucrose was collected, washed and centrifuged at
100 000 g for 30 min at 4C The resulting pellet was re-suspended in transport buffer (10 mMTris/HCl, 250 mM
sucrose, pH 7.4), and stored in aliquots at)80 C
Preparation of inside-out human erythrocyte membrane vesicles
Fresh venous blood was drawn from donors into tubes containing EDTA or heparin and processed immediately There were five donors, each of whom gave informed consent, two of northern European origin, one southern European, one Chinese, and one Sri-Lankan Membrane vesicles were prepared by a spontaneous, one-step vesicu-lation process as previously described [26–28] with minor modifications Briefly, red blood cells were washed three times with 5 vols of isotonic medium (80 mMKCl; 70 mM
NaCl; 0.2 mM MgCl2; 1 0 mM Hepes; 0.1mM EGTA,
pH 7.5) Higher concentrations of EGTA (0.5–3 mM) and high pH (8.5) interfere with the vesiculation process [26] The buffy coat and topmost cell layer were removed after each wash The packed cells were then lysed by addition to
90 vols of ice-cold solution L (2 mM Hepes and 0.1mM
EGTA, pH 7.5) and subsequently centrifuged at 40 000 g for 20 min at 4C The supernatant was removed and the pelleted ghosts were re-suspended in ice-cold solution L This step was repeated twice After the last wash, the pellets were re-suspended by addition of half the original packed cell volume of cold solution L and incubated at 37C for
30 min resulting in spontaneous formation of spectrin-actin-free vesicles [28] After incubation, the suspension was washed with solution L and the resulting pellet resuspended
in 10 mMTris/HCl (pH 7.4) The protein concentrations of the vesicle samples were determined using the BCA (bicinchoninic acid) protein assay (Pierce) Membrane vesicles were frozen and stored at)80 C until use
Trang 3Measurement of membrane vesicle sidedness
The proportion of inside-out vesicles in the membrane
preparations was assessed by determining the accessibility of
the ectoenzyme acetylcholinesterase, and the endoenzyme
glyceraldehyde-3-phosphate dehydrogenase to their
sub-strates Triton X-100 was used to disrupt the permeability
barrier and expose latent markers The determination of
enzyme activities was performed colorimetrically [29,30]
The assays were modified by exchange of all phosphate
solutions with 10 mM Tris/HCl (pH 7.4) for the assays
involving membrane vesicles prepared from human
eryth-rocytes The pH optimum of glyceraldehyde 3-phosphate
dehydrogenase activity is about 8.4 [31], but the activity in
the present study was determined at pH 7.4 to obtain
comparable conditions in the assays of sidedness and
transport Generally 30–37% of the vesicles were inside-out
Vesicle uptake studies
ATP-dependent uptake of radiolabeled cGMP or DNP-SG
into erythrocyte membrane vesicles was measured by a
rapid filtration technique [20] Thawed membrane vesicles
were diluted in buffer and 50 lg protein added to a buffer
system (55 lL final volume) containing 1mMATP, 10 mM
MgCl2, 1 0 mM creatine phosphate, 100 lgÆmL)1 creatine
kinase, 10 mM Tris/HCl (pH 7.4) and 3.3 lM [3H]cGMP
or 3 lM [3H]DNP-SG or 254 lM [3H]DNP-SG Aliquots
(20 lL) were taken from the mixture after 15 min in the case
of cGMP uptake, after 30 min with 3 lM [3H]DNP-SG
uptake, and after 45 min with 254 lM[3H]DNP-SG uptake,
diluted in 1mL of ice cold stop solution (10 mMTris/HCl,
pH 7.4) and subsequently filtered through nitrocellulose
filters (Whatman 0.2 lm pore size, presoaked overnight in
3% (w/v) bovine serum albumin The filters were rinsed
with 3 mL of ice-cold stop solution and the tracer retained
on the filter was determined by liquid scintillation counting
All transport data are presented as the difference between
the values measured in the presence of ATP and those
measured in the presence of the nonhydrolysable ATP
analogue, ATP-c-S The ATP regenerating system (10 mM
creatine phosphate, 100 lgÆml)1creatine kinase) was
pre-sent in both cases Uptake of the substrate was expressed
relative to the protein concentration of the membrane
vesicles, and all data were corrected for the amount of
radiolabelled substrate bound to the filter in the absence of
vesicle protein The substrate and inhibitor concentrations
are given in the respective figure legends Tested compounds
were added from a stock solution in the appropriate solvent
[10 mMTris/HCl (pH, 7.4), dimethyl sulphoxide or ethanol,
with the latter two solvents at a final concentration < 0.5%
v/v], identical concentrations of the vehicle being used in
control samples
Curve fitting and statistics
Data are reported as mean ± s.e.m Estimates of maximum
uptake rates and apparent dissociation constants were
obtained by least squares fits to the data using either the
solver in MicrosoftEXCEL orKALEIDAGRAPH (Synergy
Software, Reading, PA, USA) Measurements of uptake vs
substrate concentration were fitted assuming that transport
occurs as the sum of two processes each described by a Hill equation [32]:
U¼ Umaxc
n
Kn
Dþ cn; 1 n 2 ð1Þ where c is the concentration, Umaxthe maximum uptake,
Kd the apparent dissociation constant and n is the Hill coefficient, here expected to lie between 1and 2 The data were fitted to minimize the sum of squared proportional deviations,
SSE¼X
i
Ui;observed Ui;fitted
=Ui;fitted
ð2Þ fits to inhibition curves were to equations of the form:
U¼ðU0 UnoninhÞ IC
n i 50
ICni
50þ In i þ Unoninh; 1¼ ni¼ 2 ð3Þ where I is the concentration of the inhibitor, IC50 is the inhibitor concentration producing 50% inhibition of the inhibitable component, U0is the uptake in the absence of the inhibitor, Unoninhis the uptake which cannot be inhibited and niis the Hill coefficient for the inhibitor For simple competition, ni¼ 1 Unless stated otherwise, Kaleida-Graph was used to fit the data using the variances at each concentration, ri, as the weights:
SSE¼ v2¼X
i
Ui;observed Ui;fitted
=ri
ð4Þ
fits with different numbers of fitting parameters were compared using an F-test on the ratio of the variance associated with the reduction in degrees of freedom to the variance of the fit with the smaller number of degrees of freedom [33],
VR¼ðSSE2 SSE1Þ=ðd.f.2 d.f.1Þ
SSE1=d.f.1
ð5Þ where d.f is the number of degrees of freedom (¼ number of data points) number of fitting parameters) The improve-ment in fit is labelled significant if the probability from the F-test is less than 0.05 Fits with the same number of degrees
of freedom were compared with each other using the likelihood ratio:
LR¼ exp DSSE
2r2
ð6Þ where DSSE is the difference in SSE for the two fits and the noise variance was estimated as r2¼ MinSSE/d.f with MinSSEequal to the value of SSE for the best fit SDS/PAGE and Western blotting
Membrane vesicle or crude lysate proteins (5–40 lg) were separated through 7.5% (w/v) polyacrylamide and subse-quently transferred onto Hybond ECL nitrocellulose mem-branes (Amersham Biosciences) Each membrane was then incubated in blocking buffer [5% (w/v) milk powder
in 0.1% NaCl/Pi/Tris/Tween (25 mM Tris/HCl, pH 7.4,
150 mM NaCl, 0.1% Tween 20)] overnight at 4C prior
to the addition of the primary Ig (MI-1, 1 : 40 dilution;
Trang 4anti-MRP4, 1: 300 dilution) The positions of the MRP
proteins on the membranes were visualized using the enhanced
chemiluminescence horseradish peroxidase (HRP) detection
system (Amersham Biosciences) The secondary antibodies
used were the HRP-conjugated rabbit anti-(rat IgG) Ig
(1: 2000 dilution for M5I-1) or HRP-conjugated rabbit
anti-(mouse IgG) Ig (1: 2000 dilution for anti-MRP4)
Membrane proteins from the human erythrocytes were
N-deglycosylated by treatment with PNGaseF as follows:
Briefly, membrane vesicles from human erythrocytes
(40 lg) were first denatured at 100C for 10 min in the
presence of 0.5% SDS and 1% b-mercaptoethanol,
fol-lowed by incubation at 37C for 1h in the presence of
50 mM sodium phosphate (pH 7.5), 1% of the nonionic
detergent Nonidet P-40 (NP-40), and 2000 units of PNGase
F (New England Biolabs) PNGase F is an amidase which
cleaves between the innermost N-acetylglucosamine
(Glc-NAc) and asparagine residues of high mannose, hybrid and
complex oligosaccharides from N-linked glycoproteins [34]
PNGase F hydrolyzes nearly all types of N-glycan chains
from glycopeptides/proteins
Results
ATP-dependent uptake of cGMP into human
erythrocyte membrane vesicles
The rate of ATP-dependent uptake of 3.3 lM[3H]cGMP at
37C into inside-out erythrocyte membrane vesicles was
approximately constant for more than 30 min at about
1 0 pmolÆmg)1Æmin)1(Fig 1A) Uptake of [3H]cGMP in the
absence of ATP but in the presence of the nonhydrolysable
ATP analogue, ATP-c-S, was less than 5% of the uptake
in the presence of ATP The amount of [3H]cGMP taken
up by these vesicles was approximately twofold lower
(44 ± 3%, mean ± SEM, n¼ 4) when measured with
NaCl/Pi(140 mMNaCl; 3 mMKCl; 10 mMNa2HPO4; and
1.8 mMKH2PO4, pH 7.4) than with the usual low
osmol-ality transport buffer Such a difference is to be expected as
the higher osmolality should decrease the volume of the
intravesicular space
ATP-dependent uptake of[3H]cGMP was determined at
cGMP concentrations in the range 0.5–300 lM(Fig 1B,C)
To test whether the uptake occurs via a single component,
the data were fitted assuming two components each
described by a Hill equation (see Eqn 1) as shown in
Fig 1and Table 1 The data imply that there is a large
(Umax> 300 pmolÆmg)1Æmin)1), weakly cooperative (n
1.1–1.4) low affinity component with dissociation constant,
Kd2, in the range 50–85 lMand suggest that there may also
be a second, much smaller high affinity component with
Kd1, in the range 0.5–2.5 lM However, this latter
compo-nent, which may correspond to the uptake observed
previously [2,5], contributes less than 20% of the uptake
even for a low concentration, 3.3 lM, of cGMP
ATP-dependent uptake of DNP-SG into inside-out,
human erythrocyte vesicles
When inside-out erythrocyte membrane vesicles were
incu-bated at 37C with 3 lM[3H]DNP-SG, the uptake in the
presence of 1m ATP increased linearly in time for at least
Fig 1 ATP-dependent uptake of cGMP into inside-out membrane vesicles prepared from human erythrocytes (Top) Uptake of 3.3 l M
[ 3 H]cGMP was measured in the presence of 1m M ATP or the non-hydrolysable analogue ATP-cS (Middle) The variation of uptake rate with concentration of cGMP (Bottom) Haynes–Wolfe plot of the data for low concentrations In this type of plot a single, simple saturable component of uptake (Hill coefficient ¼ 1) would yield a straight line The fitted constants for the curves in (Middle) and (Bottom) are given
in Table 1 The dotted curves are drawn for a single, simple saturable component of transport (Hill coefficient ¼ 1); the dashed curves for a single component described by a Hill equation with Hill coeffi-cient ¼ 1.09, and the solid curve for two components, each described
by a Hill equation with Hill coefficients of 2 for the high affinity, low capacity component and 1.3 for the low affinity, high capacity com-ponent Data for the four highest concentrations were determined in three independent experiments from one preparation of vesicles All other data points represent at least three experiments and two vesicle preparations.
Trang 560 min while uptake when ATP was replaced by ATP-c-S
was almost negligible [12] ATP-dependent uptake of
[3H]DNP-SG in human erythrocyte vesicles was determined
over a broad concentration range (0.44–1000 lM) (Fig 2)
As for cGMP, the data for DNP-SG were analyzed using a
two component Hill equation The results of fits with several
different restrictive assumptions are shown in Table 2 The
quantitative fitting (variance ratio test) confirms, as is
obvious by eye, that the transport occurs via at least two
components To explore the range of acceptable values of
the Hill coefficient for the low-affinity component, n2, least
squares fits were obtained for specified values of n2
(Table 2) Acceptable fits (likelihood ratio¼ 0.05
com-pared to the best fit) were obtained for values of n2between
1and 1.48 Over the range from 1to 1.4, the low-affinity
dissociation constant decreases from 82 to 65 lMwhile that
for the high-affinity component varies from 0.5 to 2 lM
In agreement with previous work [10,11] the high affinity
component of DNP-SG transport in these vesicles is most
likely mediated by MRP1[12] Strong evidence in support
of this comes from the observation that the uptake rate of
3 lMDNP-SG is reduced by at least 80% by QCRL-3 [12],
an MRP1-specific conformational-dependent monoclonal
Ig [35]
To investigate the low affinity component the
DNP-SG concentration was increased to 254 lM The
inhibi-tion by QCRL-3 was then only 40 ± 5% (n¼ 6) This
result and the complete inhibition observed at low
DNP-SG concentrations suggests that there is some low
affinity transport that is not inhibited by QCRL-3 and
is thus not mediated by MRP1 On this basis, the low affinity process should account for no more than 20% of the uptake observed at 3 lM This requirement is consistent with the uptake measurements provided
n2¼ 1.4 All the data are consistent with high affinity transport via MRP1(Kd¼ 2 lM, Umax¼ 20 pmolÆmg)1Æ min)1) and low affinity, weakly cooperative transport via
a second transporter (Kd¼ 65 lM, Umax¼ 196 pmolÆ
mg)1Æmin)1 and n2¼ 1.4)
Interrelations between cGMP and DNP-SG uptake into human erythrocyte membrane vesicles
To explore the relations between cGMP and DNP-SG transport, the ability of each to inhibit transport of the other was investigated ATP-dependent uptake of 3 lMDNP-SG was not affected by the presence of cGMP at concentrations
up to 500 lM (Fig 3A) suggesting that the high affinity DNP-SG transporter, MRP1, does not transport cGMP
Table 1 Fitting parameters for the uptake of [3H]cGMP into one-step,
inside out erythrocyte membrane vesicles shown in Fig 1 The maximum
uptake rates, U max1 and U max2 , the dissociation constants, K d1 and K d2 ,
and the Hill coefficients, n 1 and n 2 are defined as indicated in Eqn (1).
The data were obtained using two different vesicle preparations As no
differences were observed between the two, the data were combined
without scaling The residual value of the sum of squared proportional
deviations, SSE (see Eqn 2), is shown for each fit For each column
except the first the variance ratio (see Eqn 5) has been calculated
rel-ative to the column immediately to the left The fit obtained with the
constraints n 1 ¼ 1and n 2 ¼ 1is not shown as the fitted value of U max1
was 0.000 These data imply (F-test on the variance ratio, P ¼ 0.0004)
that the low affinity component shows cooperativity, n 2 > 1 , and are
consistent with the presence of a high affinity component (F-test,
P ¼ 0.002), but do not specify its characteristics.
Fitted constant
Constraints
U max1 ¼ 0
n 2 ¼ 1 U max1 ¼ 0 n 1 ¼ 1 n 1 < ¼ 2
U max1 /pmolÆmg)1Æ
min)1
K d1 /l M – – 2.35 0.674
U max2 /pmolÆmg)1Æ
min)1
n 2 1.00 1.09 1.40 1.32
SSE 0.543 0.353 0.223 0.208
Fig 2 Rate of ATP-dependent uptake of [3H]DNP-SG into inside-out erythrocyte membrane vesicles The dotted curve is drawn for a single simple saturable component of uptake, the solid curve for two simple saturable components The dashed and dash-dot curves are drawn for two components each obeying a Hill equation with the constraints that
n 2 ¼ 1.4 or n 2 ¼ 2, respectively The data are plotted directly (Top) and as a Haynes–Wolfe plot (Bottom) The fitted constants are des-cribed in Table 2 These data are not consistent with a single-compo-nent of uptake, but cannot unambiguously determine the properties of two components when provision is made for the possibility that more than one substrate molecule may interact with the transporter at a time.
Trang 6This finding was further supported by the observation that
the MRP1-specific Ig, QCRL-3, produced negligible
inhi-bition of the ATP-dependent uptake of cGMP (uptake
rate for 3.3 lM cGMP with 10 lgÆmL)1 QCRL-3 was
98 ± 5% of control, n¼ 3)
On the other hand, ATP-dependent uptake of DNP-SG at
high concentrations was inhibited by cGMP (Fig 3B) The
fitted curve for uptake at 254 lM DNP-SG has two
components: a noninhibitable uptake (30 min) of 2943 ±
1 1 5 pmolÆmg)1and a component of 4110 ± 144 pmolÆmg)1
inhibitable by cGMP with an IC50of 133 ± 18 lM These
components are plausibly attributed to the high and
low-affinity components of DNP-SG transport, respectively
In order to investigate whether the cGMP transport is also
affected by increasing concentrations of DNP-SG, uptake of
3.3 lM [3H]cGMP in inside-out membrane vesicles was
measured in the presence of DNP-SG in the range of 0.5–
800 lM (Fig 3C) DNP-SG was able to inhibit all of the
cGMP transport detectable at this concentration suggesting
that it occurs via a single, DNP-SG inhibitable component
The solid curve is a plot of a Hill equation (see Eqn 3) with
IC5082 ± 2 lMand a Hill coefficient of 1.25 ± 0.02 lM
Effect of MRP inhibitors, substrates, and modulators on
cGMP uptake into human erythrocyte membrane vesicles:
A number of compounds that are known to interact with
one or more MRPs were tested for their ability to inhibit
cGMP transport (see Fig 4 and section below entitled
cGMP transport is inhibited by anion transport inhibitors,
PKC inhibitors and IBMX) MK-571, a leukotriene D4
(LTD4) receptor antagonist, which has been shown to
inhibit transport by MRP1[36,37], MRP2 and MRP3 [38]
and MRP4- [39] but not MRP5-mediated cGMP transport
[9] completely inhibited the [3H]cGMP uptake in vesicles
with an IC50value of 0.38 ± 0.01 lM(Fig 4A) suggesting
that this transport is not mediated by MRP5 cAMP
(Fig 4B) which is transported by MRP4 and MRP5 [9,13],
inhibited with IC50¼ 296 ± 26 lM The ratio of this constant to the apparent dissociation constant for cGMP, 50–80 lM(Table 1), is similar to the ratio, 6, for MRP4 [13], but is much smaller than the ratio, 380, for MRP5 [9] Glibenclamide (Fig 4C), an agent known to bind to various ABC proteins [40,41] including the sulphonylurea receptor [42,43], was effective in inhibiting the cGMP transport in human erythrocyte vesicles at micromolar concentrations Substantial inhibition was produced by methotrexate and
E217bG, established MRP4 substrates, by indomethacin which is known to inhibit transport by MRP1and MRP2 [44], and by clotrimazole (Fig 4D) an imidazole-derived antifungal agent which inhibits MRP1mediated transport [12] Imidazole, the backbone molecule of clotrimazole had
no effect Taurocholic acid, an established substrate for MRP1, MRP2, and MRP3, inhibited but only at concen-trations sufficiently high (> 200 lM, Table 3) that it may be acting in a detergent like manner
Reduced glutathione (GSH, pH 7.4), in the range of 0.5–4 mM, neither enhanced nor inhibited cGMP uptake (Table 3) This contrasts with the effect of 1–5 mMGSH to stimulate uptake of DNP-SG in human erythrocyte vesicles [12] but is consistent with the lack of effect of GSH on MRP4 and MRP5 mediated transport in transfected HEK293 cells [17]
The cationic vinca alkaloid, vincristine, and the organic anion, calcein, which are established MRP1substrates, were also tested for their ability to inhibit cGMP uptake Calcein inhibited cGMP uptake (about 25% inhibition at 100 lM
and about 60% at 300 lM) but vincristine itself had no effect even at 200 lM (Table 3) It is known that several cationic MRP1substrates, including vincristine, require GSH for their transport and that vincristine inhibits the high affinity DNP-SG transport in the presence but not in the absence of GSH [12,45,46] Thus the effect of vincristine
on the cGMP uptake was also tested in the presence of
Table 2 Fitting parameters for uptake of [3H]DNP-SG into one-step, inside out erythrocyte membrane vesicles The maximum uptake rates, U max1
and U max2 , the dissociation constants, K d1 and K d2 , and the Hill coefficients, n 1 and n 2 are as defined in Eqn (1) The data were collected in three series using different vesicle preparations To allow simultaneous fitting of all three sets of data, all data in the first set are scaled by multiplication by
AF and all data in the second by CF For fits of the two component Hill equation, there are 22 remaining degrees of freedom The one and two component fits are compared with each other using an F-test on the variance ratio (Eqn 5) The two component fit is significantly better The various constrained two component fits are compared with the fit for n 1 > ¼ 1, n 2 > ¼ 1using the likelihood ratio (Eqn 6) These data are consistent with any value of n 2 between 1 and 1.48 (LR ¼ 0.05).
Fitting constant
Constraints
U max1 ¼ 0;
n 2 > ¼ 1
n 1 > ¼ 1;
n 2 > ¼ 1
n 1 > ¼ 1;
n 2 > ¼ 1.2
n 1 > ¼ 1;
n 2 > ¼ 1.4
n 1 > ¼ 1;
n 2 > ¼ 2
Trang 71 mM GSH but no inhibition was observed Given that MRP1-mediated transport of calcein in whole cells can be modulated by the level of GSH present [47], the effect of calcein on cGMP uptake into erythrocyte membrane vesicles was also tested in the presence of 1mMGSH but
no additional inhibition was observed (Table 3)
These results are all consistent with cGMP transport via MRP4 while the strong inhibition produced by MK-571 and the relative potency of cAMP appear to be incompati-ble with transport via MRP5
CGMP transport is inhibited by anion transport inhibitors, PKC inhibitors and IBMX
Because substrates for MRPs are often organic anions, inhibitors that block ion transport were tested (Table 4) The anion transport inhibitors frusemide, niflumic acid, phloridzin, SITS and probenecid all reduced the rate of cGMP uptake (Table 4) By contrast the potassium channel blockers, 4-aminopyridine, tetraethylammonium chloride and CsCl, had no observable effect though BaCl2 did (Table 4) Verapamil, a calcium channel blocker, an inhi-bitor of P-glycoprotein, and a general though weak inhiinhi-bitor
of MRPs in vesicular drug uptake studies, reduced cGMP transport in the presence or absence of 1mM GSH Two protein kinase C inhibitors, staurosporine and Ro 31–8220 [48], were also tested for their ability to block cGMP transport in human erythrocytes Staurosporine has recently been shown to bind directly to several ABC transporters [49]
in addition to preventing phosphorylation of these trans-porters in intact cells [50] Staurosporine at 10 lM com-pletely inhibited the cGMP uptake while Ro 31–8220 at
10 lMshowed only weak inhibition Forskolin, an activator
of adenylyl cyclase, inhibited while its inactive analogue, 1,9-dideoxyforskolin, had no effect at the same concentra-tion IBMX which is structurally related to cGMP and currently used as a nonspecific phosphodiesterase inhibitor, inhibited transport All of these effects are compatible with cGMP transport by a member of the MRP family
Immunodetection of MRP4 and MRP5 proteins
in human erythrocytes and COR-L23/R cells
To identify candidate proteins that could possibly mediate the cGMP transport, immunoblot analysis was performed
on membrane vesicles from human erythrocytes using monoclonal antibodies against MRP5 [18] and MRP4 [19] The anti-MRP5 Ig, M5I-1, specifically detected an intact band at 190 kDa which shifted to 160 kDa after treatment with peptide N-glycosidase F (PNGaseF) (Fig 5A) suggest-ing that MRP5 is N-glycosylated A protein with the same apparent molecular mass was also detected in doxorubicin-resistant MOR/R and COR-L23/R lung tumour cells but at greatly reduced level in the doxorubicin-sensitive COR-L23/
P lung tumour cells (Fig 5B) The anti-MRP4 Ig detected an intact band at 170–180 kDa in human erythrocytes and MOR/R cells but not in COR-L23/R cells (Fig 5B) Discussion
It is now well recognized that inside-out membrane vesicles prepared from human erythrocytes can take up both
Fig 3 Effect of cGMP on the DNP-SG transport in human
erythro-cytes and vice versa (Top) ATP-dependent uptake of 3 l M [ 3
H]DNP-SG (30 min at 37 C) was not affected by cGMP at concentrations up
to 500 l M (Middle) ATP-dependent uptake of 254 l M [ 3 H]DNP-SG
(30 min at 37 C) was partially inhibited by cGMP The fitted curve
corresponds to two components: a noninhibitable component of
2943 ± 115 pmolÆmg)1and a component of 4110 ± 144 pmolÆmg)1
inhibited by cGMP with an IC 50 of 133 ± 18 l M (mean ± SEM).
(Bottom) ATP-dependent uptake of 3.3 l M [3H]cGMP (15 min at
37 C) was inhibited by DNP-SG as a single component described by
a Hill equation with U 0 ¼ 123 ± 1 pmolÆmg)1, IC 50 ¼ 82 ± 2
pmolÆmg)1, and a Hill coefficient of 1.25 ± 0.02 (v2¼ 208) The fit of
the Hill equation was significantly better (variance ratio test, 17 data
points, three parameters in the Hill equation, P ¼ 0.003) than the
fit to a simple competition curve (U 0 ¼ 135 ± 1 pmolÆmg)1,
IC 50 ¼ 47 ± 1pmolÆmg)1, v2¼ 395).
Trang 8glutathione-conjugates, such as DNP-SG, and cyclic
nucleo-tides, e.g cGMP, by rapid ATP-dependent transport
processes In the present study, uptake of cGMP is shown
to consist primarily of a low-affinity component with a
maximum uptake (Umax) of 300–400 pmolÆmg)1Æmin)1and
a dissociation constant (Kd) in the range of 50–82 lMand
possibly also a second high affinity component of uptake
contributing less than 20% of the total trasnport even at low
concentrations However, MK-571, glibenclamide,
DNP-SG, clotrimazole and cAMP all inhibit this uptake as if
there were only a single component of transport (Figs 3C
and 4) If present, the high-affinity component may
correspond to the high-affinity transport previously
repor-ted In those studies [2,5] ATP-dependent cGMP uptake
into inside-out membrane vesicles from human erythrocytes
was found to have two components, a high affinity uptake
with Umax1¼ 0.2–0.4 pmolÆmg)1Æmin)1 and Kd1¼ 2.4– 4.7 lM and a low affinity uptake with Umax2¼ 1.6 pmolÆmg)1Æmin)1 and Kd2¼ 170 lM The maximum up-take rates in these studies were very low, being just over threefold higher than the background [2] The maximum uptake rate in the current study is two orders of magnitude higher The reasons for this remarkable difference are unclear though there may be several factors involved These include different osmolalities of the solutions used to measure uptake, differences in the methods of vesicle preparation, and possibly even differences in the profile of transporters present on the red cell membranes from different donors
In the present study, the vesicles were resealed and assayed in low osmolality solution This contrasts with the previous study where the vesicles were resealed at low
Fig 4 Inhibition of uptake of 3.3 l M cGMP by (A) MK-571, (B) cAMP, (C) glibenclamide and (D) clotrimazole (A) Inhibition by MK-571 The curve is the best fit assuming simple competition, U 0 ¼ 130 ± 2 pmolÆmg)1, IC 50 ¼ 0.38 ± 0.01 l M , v 2
¼ 95 The Hill equation provides a closer
fit with, U 0 ¼ 120 ± 2 pmolÆmg)1, IC 50 ¼ 0.48 ± 0.02 l M , n i ¼ 1.10 ± 0.02 and v 2
¼ 70, but the improvement is not significant (F ¼ 2.7,
P ¼ 0.13) The best fit allowing for noninhibitable uptake assigns a negative value ( )1.4 pmolÆmg )1 ) to the noninhibitable component (B) Inhibition by cAMP The curve shows the best fit for simple competition with U 0 ¼ 108 ± 5 pmolÆmg)1, IC 50 ¼ 296 ± 26 l M , v 2
¼ 1 1 Fits
of the Hill equation and of simple competition plus a noninhibitable component of uptake are almost superimposed on that shown (and improvements in fit were not-significant with F and P-values for the variance ratio relative to simple-competition of 0.03 and 0.86, and 0.24 and 0.64, respectively) (C) Inibition by glibenclamide The curve is the best fit for simple competition U 0 ¼ 80 ± 1pmolÆmg)1, IC 50 ¼ 2.8 ± 0.1 l M ,
v 2
¼ 250 Fits of the Hill equation and of simple competition plus a noninhibitable component of uptake did not produce significant improvements
in the fit (F and P-values 3.26 & 0.12 and 5.0 & 0.07, respectively) The best fit for the noninhibitable component was 3.4 ± 0.3 pmolÆmg)1 (D) Inhibition by clotrimazole The curve is the best fit for simple competition U 0 ¼ 97 ± 1pmolÆmg)1, IC 50 ¼ 24 ± 1 l M , v2¼ 86 Fits of the Hill equation and of simple competition plus a noninhibitable component did not produce significant improvements in the fit (F and P-values of 4.4
& 0.1and 4.7 & 0.1, respectively The best fit value of the Hill coefficient was less than 1(0.72).
Trang 9osmolality but assayed in NaCl/Pi with an osmolality
10–100-fold higher To check these solutions as possible
influencing factors, cGMP uptake in the current study was
also measured using solutions as employed in the previous
studies Under these conditions, uptake rates were lower,
but only by a factor of two, i.e too small an effect to explain
alone the discrepancies in results between the two studies
Another possible explanation for the discrepancy is the
presence of traces of calcium in the previous studies It was
shown there that calcium can inhibit cGMP transport with
an IC50of about 40 lMand the inclusion of 100 lMEGTA
was found to increase uptake rates by 100% [4] Even so no
chelators were included in most of those studies either
during vesicle preparation or during uptake measurements
The vesicle preparations used in the present work were
produced by a one-step spontaneous vesiculation method in
the presence of 100 lMEGTA [26–28]
Further differences relate to the percentage of inside-out
vesicles generated The previous study used the procedure of
Steck and Kant [30] which yields a much higher percentage
(typically > 60%) of inside-out vesicles than are routinely
produced by spontaneous vesiculation (usually 30–37%)
However, these differences would be expected to produce
lower uptake rates in the vesicles generated by spontaneous
vesiculation, not higher rates as observed here Whether
there are inhibitory elements on the vesicle membranes that
are stripped off more effectively by spontaneous
vesicula-tion or stimulatory elements removed in the lengthy Steck
and Kant procedure remains to be determined Certainly it
has been proposed that the spontaneous vesiculation
process may remove restrictive links with cytoskeletal
elements [28] allowing more lateral mobility and thus the
possibility of alterations in associations between membrane
proteins The additional possibility that different donors possess different profiles of transporters on their red cell membranes is currently being explored
In contrast to uptake of cGMP, ATP-dependent uptake
of DNP-SG into inside-out erythrocyte membrane vesicles
in the current study clearly possesses more than one component This has been noted also by Akerboom et al [7] and Pulaski et al [10] The observations are consistent with the presence of two components, one, a low-capacity
Table 3 Effect of MRP substrates, inhibitors and modulators on the
ATP-dependent uptake of [3H]cGMP by inside-out human erythrocyte
membrane vesicles Data represent mean ± SEM of n experiments The
control uptake of [3H]cGMP (addition of dimethylsulfoxide only) for
indomethacin, methotrexate and E 2 17bG was 77.3 ± 5.2 pmolÆmg)1.
The control uptake for the remaining drugs was 136.5 ± 2 pmolÆ
mg)1.
Compound
Concentration (l M )
[ 3 H]cGMP uptake (% control) n
1000 108.3 ± 7.1 6
2000 100.1 ± 1.9 3
4000 98.0 ± 1.2 3 Vincristine 100 106.3 ± 7.6 3
200 100.6 ± 3.2 3 + 1 m M GSH 100 116.4 ± 1.1 3
+ 1 m M GSH 100 74.4 ± 1.8 3
Indomethacin 20 95.9 ± 3.7 3
Methotrexate 275 25.2 ± 2.8 3
Taurocholic acid 200 80.1± 2.1 3
Table 4 Effect of ion channel inhibitors on the ATP-dependent uptake
of [3H]cGMP by inside-out human erythrocyte membrane vesicles Data represent mean ± SEM of n experiments The control level of [3H]cGMP uptake with dimethylsulfoxide (n ¼ 16) for furosemide, SITS, probenecid, staurosporine and Ro 31–8220 was in the range of 77–82 pmolÆmg)1protein The control level of [ 3 H]cGMP uptake with ethanol (n ¼ 18) for phloridzin, niflumic acid, and verapamil was in the range of 90–105 pmolÆmg)1 protein The control level of [ 3 H]cGMP uptake for the remaining drugs was in the range of 129–
135 pmol mg)1protein.
Compound
Concentration (l M )
[3H]cGMP uptake (%control) n
Anion channel inhibitors Frusemide 0.5 95.3 ± 2.3 6
5 37.1± 2.1 8
10 19.4 ± 2.6 6
20 6.6 ± 0.9 4
50 6.9 ± 1.3 9 Niflumic acid 11 05.2 ± 2.1 4
5 45.6 ± 3.7 6
10 21.4 ± 1.5 6
20 5.4 ± 0.5 3
50 5.6 ± 1.1 9 Phloridzin 11 09.8 ± 3.3 5
5 99.9 ± 4.7 5
10 75.2 ± 4.2 7
50 28.3 ± 2.3 10
Probenecid 200 11.3 ± 0.7 3
400 8.1± 1.1 3 Cation channel inhibitors
1000 47.9 ± 1.6 3
1000 102.4 ± 3.1 3 4-Aminopyridine 500 87.7 ± 4.6 3 Tetraethylammonium Cl 10000 87.3 ± 3.4 3 Verapamil 50 66.8 ± 1.7 3 Verapamil + 1m M GSH 50 71.3 ± 3.5 3 PKC inhibitors
Staurosporine 10 2.9 ± 0.7 3 Ro31–8220 10 74.2 ± 2.3 3 Various
Colchicine 50 98.4 ± 3.0 8 Imidazole 100 94.1 ± 0.8 3 Dideoxyforskolin 100 95.3 ± 2.2 3 Forskolin 100 66.7 ± 1.6 3
Trang 10high-affinity component (Kd 1–10 lM) that predominates
at concentrations below 5 lM and another low-affinity
component (> 100 lM) that is dominant at high
concen-trations
The high-affinity component of DNP-SG transport,
identified previously as being mediated by MRP1[10–12],
appears not to be involved in transport of cGMP (see
below) However, the low affinity component of DNP-SG
transport is inhibited by cGMP and may well be the
transporter that mediates low-affininty cGMP uptake
DNP-SG can indeed inhibit cGMP transport Furthermore,
the apparent dissociation constant for DNP-SG uptake,
kd2, and the IC50 for DNP-SG-mediated inhibition of
cGMP uptake are approximately equal, i.e 65 lM
com-pared with 82 lM Though the two values for cGMP uptake
and for cGMP inhibition of DNP-SG uptake differ, i.e 50–
82 lM compared with 133 lM, it remains possible that a
single transporter accounts for both uptakes as the
inter-action between cGMP and DNP-SG is likely to be more
complex than simple competition Further evidence for
greater complexity is provided by the curve fits with Hill coefficients greater than 1
As a wide variety of MRP transport inhibitors block cGMP and DNP-SG transport (Table 3 and Fig 4), members of the MRP family are very plausible candidates for the transporters mediating ATP-dependent uptake of cGMP and DNP-SG into inside-out erythrocyte vesicles MRP1and MRP5 have previously been detected on the membranes of human erythrocytes [8,9] MRP1is known to mediate the high-affinity transport of DNP-SG [10–12] and MRP4 and MRP5 are known to transport cGMP [9,13,17]
In the present study in addition to confirming the presence
of MRP1and MRP5 in erythrocyte membranes, we have detected the presence of MRP4 (see Fig 5) Although no cross-reactivity tests have been reported for anti-MRP4, the present finding that the anti-MRP4 Ig does not detect an immunoreactive band in COR-L23/R cells which express MRP1and MRP5 suggests that this Ig does not cross-react with MRP1or MRP5
Several lines of evidence show that MRP1is most unlikely to be the transporter responsible for the transport
of cGMP It has already been shown that cGMP does not inhibit transport of LTC4, an established high-affinity MRP1substrate [4]; cGMP does not inhibit MRP1 -mediated transport of DNP-SG; and, verapamil inhibits cGMP uptake without any requirement for GSH (this study) while GSH is needed for its inhibition of MRP1-mediated transport [12,51] Most convincingly, uptake of 3.3 lMcGMP is unaffected by the presence of the confor-mation-dependent monoclonal Ig against MRP1, QCRL-3,
at 10 lgÆmL)1 which has been shown to block MRP1 mediated high-affinity uptake of DNP-SG [12]
Both MRP4 and MRP5 have been shown to transport cGMP, though the apparent dissociation constants have proven controversial The original reports indicated relat-ively high-affinity trasnport with Kmvalues in the range of 2–10 lM [9,13], but the transport is clearly much lower affinity in HEK293 cells transfected with MRP4 or MRP5 [17] To examine further the possible role of the MRPs in erythrocytes, several established MRP substrates, inhibitors, and modulators were tested for their ability to block the [3H]cGMP uptake into inside-out vesicles MK-571, a leukotriene receptor antagonist, inhibited the cGMP trans-port with an IC50value of 0.38 ± 0.01 lM This appeared
to be the most potent of the inhibitors tested in the present study MK-571has been shown to inhibit MRP1, MRP2, MRP3 and MRP4 mediated transport [9,36,38,39] but has been reported to have no affect at concentrations of up to
50 lMon cGMP transport attributed to MRP5 [9] Methotrexate and E217bG were found to inhibit the cGMP uptake in human erythrocyte vesicles with E217bG
at 65 lM inhibiting about 90% and methotrexate inhi-biting about 75% at 275 lM and completely at 375 lM These compounds are established MRP4 substrates with
Km values around 220 lM for methotrexate [19,39], and
30 lM for E217bG [39,3,4] At present, methotrexate and
E217bG appear to be substrates of MRP4 [13,39] and not
of MRP5
cAMP, which has been shown to be transported by both MRP4 and MRP5 [9,13], inhibited all of the cGMP uptake with an estimated IC50value of 315 ± 70 lM The ratio of this value to the apparent K value for cGMP, about 5, is
Fig 5 Immunodetection of MRP4 and MRP5 in human erythrocytes
and COR-L23 cells Inside-out membrane vesicles prepared from
human erythrocytes, COR-L23/P and COR-L23/R cells, and crude
lysates from MOR/ADR cells were size fractionated on 7.5% SDS/
PAGE, blotted and immunostained with (A) M 5 I-1mAb, and
(B) anti-MRP4 mAb, detecting MRP5 and MRP4, respectively.
Membranes (40 lg) from human erythrocytes were also treated with
PNGase F to remove the N-glycans and then immunostained with
M 5 I-1(A) The amount of protein loaded per lane is indicated at the
bottom of each blot Arrows mark the immunodetected band for
MRP4 and MRP5 Doxorubicin-resistant lung adenocarcinoma cell
line MOR/R was used as a positive control; doxorubicin-sensitive
human large-cell lung tumour cell line COR-L23/P was used as a
negative control.