Download The Inability of the Mouse mdr2 Gene to Confer

(CANCER RESEARCH54, ‘ts92-4@t98,
September 15. 19941
The Inability of the Mouse mdr2 Gene to Confer Multidrug Resistance Is Linked to
Reduced Drug Binding to the Protein1
Ellen Buschman and Philippe Gros2
Department of Biochemistry. McGill University. Montreal, Quebec. Canada H3G I Y6
ABSTRACT
The mouse mdr gene family is composed of three members designated
indri, mdr2, and mdr3. A full-length mdr2 complementary DNA clone
has been Introduced
in an ampliftable
eukaryotic
expression
vector
(pEMC2b1)whichdirectsamplification
andoverexpression
of a bids
tronic mdr2-dihydrofolate
selection
of transfected
reductase
mutant
mRNA after stepwise methotrexate
dihydrofolate
ovary DUK cells. Independent
reductase
cell clones expressing
Chinese
hamster
low to high amounts
of mdr2 cellular mRNA and Mdr2 protein in their membrane fraction
could be obtained by this selection procedure. Comparison of drug sur
vival characteristics
of cell clones expressing similar amounts of either
Mdrl or Mdr2 proteins revealed that Mdrl but not Mdr2 could confer
readily detectable levels of coichicine or vinblastine resistance. Labeling
experiments using membrane-enriched
fractions and a photoactivatable
analogue of ATP showed that the Mdr2 protein was properly inserted in
the membrane of transfected cells and could bind this ligand with an
apparent affinity similar to that of Mdrl. However, labeling studies with
the photoactivatable
drug analogue lodoarylazidoprazosin
showed consid
erably reduced binding of this ligand to Mdr2 as compared to Mdrl. Our
findings demonstrate
that Mdr2 cannot confer drug resistance
and sag
gest that this inability is linked to reduced drug binding to the Mdr2
protein.
INTRODUCTION
MDR3 in cultured cells in vitro and tumor cells in vivo is caused by
the overexpression of P-gps (1). P-gps have been shown to bind AlT
(2, 3) and drug analogues (4, 5) and to possess ATPase activity
(reviewed in Ref. 6), and are believed to function in resistant cells as
energy-dependent drug-efflux pumps that prevent intracellular drug
accumulation (1). P-gps are encoded by a small family of homologous
genes, termed mdr or pgp, which include two members in humans.
[MDRJ
and MDR2
(also
known
as MDR3;
Refs.
7—9)J,and three
members in mice [mdrl, mdr2, and mdr3 (10—13)].Predicted amino
acid sequence analyses indicate that P-gps are formed by two se
quence homologous halves, each composed of 6 putative TM domains
and a NB fold. P-gps show a very high degree of inter- and intraspe
cies sequence homology, with 75—85%identity among the 3 mouse
proteins. The three mouse mdr genes arose from two successive gene
duplication events, the last one creating mdrl and mdr3, which share
greater homology to one another than either do to mdr2 (12, 14).
Despite this high degree of sequence conservation, striking functional
differences have been detected between individual mdr genes in
transfection experiments. Mouse mdrl and mdr3 and human MDRJ
can directly confer drug resistance to otherwise drug-sensitive cells in
transfection experiments (12, 15), while mouse mdr2 (1 1, 16), and
human MDR2 apparently cannot (17).
Although the normal physiological role of individual human and
mouse P-gps remains unclear, their patterns of RNA and protein
expression are restricted in an organ- and cell-specific fashion. At the
cellular level, P-gps are generally expressed at the apical pole of
secretory epithelial cells, suggesting that they may function as trans
membrane transporters at these sites (1, 18). Human MDR1 is ex
pressed at highest levels in the adrenal gland, epithelia of the kidney,
jejunum, colon, and endothelial cells of the blood brain barrier,
whereas human MDR2 is expressed almost exclusively in liver (19—
23). In normal mouse tissues, mdrl is most strongly expressed in the
pregnant uterus, adrenals, placenta, and kidney, whereas mdr3 is
mostly expressed in intestine and lung (24). RNA hybridization cx
periments with gene-specific mdr probes have also identified liver as
the major site of mdr2 RNA expression (24). Recently, we have used
isoform specific anti-P-gp antibodies to show that (a) two P-gp
isoforms (Mdr2 and Mdr3) are expressed in liver with Mdr2 being the
major species, and (b) P-gp expression is highly polarized in this
tissue and restricted to the canalicular side but not the sinusoidal side
of the bile canaliculus (25). Furthermore, photolabeling studies with
the drug analogue IAAP suggested that Mdr3 but not Mdr2 was
capable of binding the drug analogue in canalicular membrane vesi
des (25). Although the precise nature of the substrate(s) transported
by Mdr2 in CMV remains unknown, the analysis of mutant mice
bearing a homozygous null mutation at mdr2 suggests that it may
participate in the transport of PC across the canalicular membrane
(26).
The inability of mouse Mdr2 and its human MDR2 homologue to
confer drug resistance and therefore to carry out drug transport has
also been addressed in transfected mammalian cells. In human BRO
cells, high level expression of MDR2 failed to confer drug resistance
with daunorubicin efflux identical to that of control drug-sensitive
cells (17). Likewise, introduction of the mouse mdr2 cDNA into
drug-sensitive CHO LR73 cells failed to yield drug-resistant colonies
upon
direct
plating
of
the
transfected
cells
in medium
containing
colchicine; DHFR, dihydrofolate reductase; CHO, Chinese hamster ovary cells; DUK,
CHO DHFR cell line DUKX-B1; EMC, encephalomyocarditis virus; IAAP, iodoaryla
either COL or ADM (1 1, 16). Transfection of CHO cells with chi
meric mdrl/mdr2 cDNA molecules created by exchanging homolo
gous domains of either protein showed that NB1 and NB2 of mdr2
could functionally complement the drug resistance function of Mdrl,
while segments overlapping the predicted TM-associated domains
could not (16). Since genetic (27—31)and biochemical analyses (32—
36) have shown that TM domains of P-gp are responsible for drug
binding, these results are in agreement with the proposition that Mdr2
transport, by a mechanism similar to Mdrl, different classes of sub
strates such as biliary PC, as suggested by Smit et a!. (26).
In the present study, we have attempted to understand the molecular
basis underlying the functional differences detected between mdrl
and mdr2. For this, we have used a novel expression system which
zidoprazosin; MEM, minimal essential medium; MTX, methotrexate; NB, nucleotide
does
Received 5/3 1/94; accepted 7/19/94.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
I This
work
was
supported
by grants
from
the
Medical
Research
Council
of Canada
(P. 0.). P. G. is supported by a fellowship from the Natural Sciences and Engineering
Research Council of Canada and is an International
Hughes Medical Institute.
2 To
whom
requests
for
reprints
should
be addressed,
Research Scholar of the Howard
at Department
of Biochemistry,
McGill University, Montreal, Quebec, Canada H3G 1Y6.
3 The
abbreviations
used
are:
MDR,
multidrug
resistance;
ADM,
Adriamycin;
COL,
not
rely
upon
MDR
drug
selection
to achieve
high
level
binding; P-gp, P-glycoprotein; SDS, sodium dodecyl sulfate; TM, transmembrane; VBL,
vinblastine; PC, phosphatidylcholine; eDNA, complementary DNA; PBS, phosphate
P-gp
expression to express high and equivalent amounts of Mdrl and Mdr2
in membrane fractions of transfected cells. The ability of the two Mdr
buffered saline; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
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DRUGBINDINGTO Mdr2
isoforms to confer drug resistance and to bind drugs as well as Al?
photoactivatable analogues was assessed.
MATERIALS
described previously (3). Purified membrane fractions were further isolated by
ultracentrifugation
AND METHODS
mdr-transfected
Cell Clones.
0.1 M NaCI, and 0.01
A full length mdr2 cDNA (nucleotides
200—
4359) (11) was cloned into the blunt-ended EcoRI site of the mammalian
expression vector pEMC2b1 (generous gift of R. Kaufman, Genetics Institute,
Cambridge, MA) and introduced as a calcium phosphate coprecipitate (37) into
drug-sensitive
mutant
Chinese
hamster
(150,000
X g for 3 h at 4°C) on a discontinuous
density
gradient of 60, 45, 35, and 30% sucrose. Membrane vesicles present at the 35
and 45% interfaces were collected, washed, and stored at —80°C
in 0.1 MTris,
ovary cells (DUK cells) bearing
a null
concentration
(Bio-Rad).
M EDTA,
pH 7.5 containing
Protein
Western Blotting. SDS-PAGE was performed according to standard pro
tocols (43). Briefly, 12.5—50@g
of purified membrane proteins were solubi
lized in 2 x Laemmli sample buffer for 5 mm at room temperature before
loading onto 7.5% polyacrylamide
mutation at the DHFR (dihydrofolate
reductase) locus. The pEMC2b1 vector
is identical to the pMT2 (38) vector, except for an EMC virus translation
40% glycerol.
was determined using an amido black-based commercial assay
gels. After electrophoresis,
ibrated in transfer buffer (20% methanol-0.76
gels were equil
M glycine-2.5
and proteins were transferred to nitrocellulose
mM Tris, pH 8)
sheets by Western blotting (0.5
reinitiation sequence positioned downstream of the mdr2 cDNA and upstream
the MTX resistance gene DHFR. Stable populations of transfectants were
A, 100 V, at 4°C)for 1—3
h. The blots were treated to prevent nonspecific
isolated
binding
after selection
bonucleotides
protocol
in a-MEM
and containing
described
culture
medium
10% dialyzed
previously
lacking
fetal bovine
(39). Mass populations
ribo- and deoxyri
serum
according
of cell clones
to a
surviving
this initial selection were harvested as pools 10 days after transfection, ex
panded in culture, and subjected to further stepwise selection in the same
medium containing 0.02, 0.04, and 0.06 ,.@M
MTX. Individual colonies growing
in 0.06 p.M MTX were harvested, expanded in culture, and frozen in 90% fetal
calf serum and 10% dimethyl sulfoxide. Drug-sensitive DUK or LR73 (40)
cells,
used as negative
controls
in all experiments,
by incubation
overnight
at 4°C in 10 mM Tns,
which recognizes
all P-gp isoforms
with goat anti-mouse
antibody
M NaCl
(44), for 1 h at room temperature,
conjugated
to alkaline
and then
phosphatase.
Specific
antigen-antibody complexes were revealed by incubation with 5-bromo-4chloro-3-indoyl
phosphate
Immunoprecipitation.
p-toluidine
and nitro blue tetrazolium
(Bio-Can).
Control and mdr-transfected cells (1.5 X 106)
growing in 60-mm dishes were metabolically
and all other cell clones
pH 8-0.15
0.02% Tween 20-1% bovine serum albumin,followed by incubationwith a
1:300 dilution of mouse anti-P-gp monoclonal antibody C219 (Centocor),
labeled for 12 h at 37°C in
were grown in a-MEM supplemented with 10% fetal calf serum, glutamine (2
a-MEM lacking methionine (Flow Laboratories), supplemented with 10%
mM), penicillin (50 units/ml) and streptomycin
dialyzed fetal calf serum, L-glutamine (2 mM), and L-[35S]methionine at 35
@Ci/ml(specific activity, 1000 Ci/mmol; Amersham). Labeled cells were
indicated.
mdrl
Multidrug-resistant
(clone
control
1-1) or mdr3 (clone
(50 p@g/ml),unless otherwise
cell lines expressing
3-5) were maintained
either wild type
in medium
washed
containing
twice with PBS, harvested
with a Pasteur pipet, solubilized
bation
levels of a chimeric
SDS, and sonicated (3 X for 20 s) on ice. Next, 0.8 ml of a solution containing
Mdr2 introduced
Mdrl/Mdr2
(clone
1-2) consisting
of the linker domain of
in Mdrl, was grown in medium containing
ng/ml (16). Finally,
the mdrl-transfected
clone EX4N-7,
which
for 5 mm at 0°C in 0.2 ml of buffer
containing
by incu
VBL at 50 ng/ml (28). A multidrug-resistant cell clone stably expressing high
50 mM Tris pH 7.5-1%
ADM at 100
1.25% Triton X-100, 200 mM NaCI, and 50 mM Tris (pH 7.5) was added and
expresses
the mixture was flushed
low
amounts of Mdrl protein, was maintained in medium lacking MDR drugs but
containing Genetycin (0.5 mg/ml) (41).
gently
tamed protease inhibitors (2
pepstatin;
Sigma).
through a 26-gauge
needle.
All buffers
con
@g'mltrasylol, 5 @tWmlleupeptin, 0.04 @.tg/ml
The solution
was clarified
by centrifugation
at 15,000
X g
Northern Blotting Experiments. Total cellular RNA was prepared by
for 15 mm at 4°Cand immunoprecipitation with the anti-P-gp monoclonal
homogenizing transfected and control cells in a solution containing guani
dinium hydrochloride (6 M), followed by serial ethanol precipitations and
complexes were purified using protein A-Sepharose (Pharmacia); washed
phenol/chloroform
extensively
analysis,
extractions,
as described
previously
10 @.tgof each RNA sample [denatured
(14).
For Northern
antibody
C219
with
150 mr@i NaCl,
in 7 X standard saline-citrate
(1:100
dilution)
a solution
50 mM Tris
was carried
containing
(Boerhinger-Mannhein);
and resolved
amide gels, as described
above.
transferred onto Gene Screen Plus (DuPont New England Nuclear) by capillary
blotting. Membranes were baked for 2 h at 70°C,prehybridized, and hybrid
ized sequentially in a solution containing 1 M NaCI, 1% SDS, and 10% dextran
Kodak XAR film.
sulfate
at 65°C for 16 h each.
citrate)-7.5%
The mdr2-specific
formaldehyde
hybridization
for 15
2276 (14). The hybridization
108
was labeled
with [a-32P]dATP
X-100,
bovine
by SDS-PAGE
The gels were fixed,
0.03%
serum
SDS,
albumin
on 7.5% polyacryl
dried, and exposed
to
PhotoafTinity Labeling. Purified membrane fractions from control and
mdr-transfected cells were incubated with IAAP (specific activity, 2000 Cu
was a
mmol; DuPont New England Nuclear) and crosslinked
described
previously
(35). For AlT-labeling,
100
with UV light, as
@.tgof purified
membrane
extracts were incubated with [a-32Pj8-azidoadenosine-5'-triphosphate
(Dupont,
of
Ic activity,
@pm/p@gDNA. The blot was washed to a final stringency
of
Labeled P-gps were recovered by immunoprecipitation with C219 and ana
Nuclear)
0.5 x standard
saline
by random primer extension
citrate,
1% SDS
at 65°C and exposed
film for 24 h at —80°Cwith an intensifying
Drug Survival
to a specific
to Kodak
2—10 Ci/mmol;
ICN
Biomedicals),
as described
(specif
activity
New England
5 x
probe
Triton
1869—
probe
410-base pair Hinfi fragment of clone A-DR29 including nucleotides
0.1%
(pH 8), and 5 mg/ml
mm at 65°C]were fractionated in formaldehyde-containing agarose gels and
(1 X SSC = 0.15 M NaCl, 0.015 M sodium
out for 16 h at 4°C. Immune
previously
(3).
lyzed by SDS-PAGE exactly as described above. Gels were fixed, dried, and
exposed to Kodak XAR films with an intensifying screen (Kronex, Dupont de
XAR
screen.
Assay. A modification of a cell survival assay (42) based
Numours) at —70°C
for 1—2
weeks.
on sulforhodamine B staining of total cell protein was used. Briefly, 5 X i03
drug-sensitive
DUK or LR73 control
cells or mdr-transfected
cells were plated
in 96-well titer plates in complete medium containing increasing concentra
tions of VBL or COL and incubated for 72 h at 37°C.Cells were then washed
RESULTS
once in ice-cold PBS, fixed in 17% trichloroacetic acid in PBS for 1 h at 4°C,
and then washed extensively
in tap water. Cellular proteins were stained with
As opposed to its mdrl and mdr3 homologues, introduction of the
mouse mdr2 cDNA in LR73 CHO cells fails to yield drug-resistant
colonies on plating the transfected cells in cultured medium contain
ing COL or ADM (11, 16). Although these experiments suggest that
mdr2 cannot confer MDR, appropriate expression of the full-length
protein in the membrane fraction of these cells needs to be demon
strated to sustain this conclusion. Therefore, the aim of the present
study was to express the Mdr2 protein in stably transfected cells,
initiate a biochemical analysis of the protein, and try to identify the
basis for its apparent inability to confer drug resistance. Initial exper
iments using mdr2 cDNA inserted in the eukaryotic expression vector
pMT2 and cotransfected with indicator plasmid pSV2neo produced
stable G41& colonies which expressed either no or only very low
a solution of 0.4% sulforhodamine
B in 1% acetic acid for 15 mm at room
temperature, followed by 4 washes with 1% acetic acid to remove excess stain.
After the plates were dried, the stain was dissolved
in 10 mM Tris (pH 9.0) and
quantification was carried out using an automated enzyme-linked immunosor
bent assay plate reader (Bio-Rad Model 450) set at 490 nm. The relative
plating
efficiency
of each
clone
was calculated
by dividing
the absorbance
observed at a given drug concentration by the absorbance detected in the same
clone in medium devoid of drug and is expressed as a percentage. The drug
dose required
to reduce
plating
efficiency
of each clone
by 50%, was also
calculated.
Isolation
of Membrane
tured cell clones
Fractions.
Crude
were prepared by homogenizing
membrane
extracts
cells in hypotonic
from
cul
buffer as
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DRUG BINDING TO Mdr2
amounts of the Mdr2 protein, insufficient for biochemical analysis
(data not shown). To increase the level of protein expression in
transfected cells, we turned to the eukaryotic expression vector
pEMC2b1. The expression cassette of pEMC2b1 is similar to that of
pMT2 (38), except that the mdr2 cDNA is fused to DHFR cDNA with
an EMC virus translation reinitiation sequence separating the two
cDNAs. Transfection of such constructs into DHFR mutant CHO
(DUK) cells, followed by selection in increasing concentrations of
MTX, yields cell clones producing very high levels of bicistronic
recombinant DHFR mRNAs allowing high level expression of the
foreign protein encoded by the introduced cDNA.
The pEMC2b1-mdr2 construct was introduced into DUK cells, and
transfected cell clones were selected first in a-MEM lacking ribo- and
deoxyribonucleotides, followed by stepwise selection in increasing
concentrations of MTX up to 0.06 ,[email protected] independent clones
were isolated, expanded in culture, and examined for the presence of
mdr2 mRNA and protein expression. Northern blotting analysis with
an mdr2-specific hybridization probe of a representative cell clone
fairly abundant (Fig. 1B; Lanes 2 and 6). Nevertheless, the amount of
Mdr2 protein present in the highest P-gp-expressing clone was still
considerably lower (10—20%)than the amount of P-gp expressed in
multidrug-resistant cell clones transfected and overexpressing either
wild type Mdrl or Mdr3 proteins (Fig. 1C; Lanes 2 and 6, respec
tively) or the Mdrl-Mdr2 chimera (Fig. 1B; Lane 11). In agreement
with our previous analysis of Mdr2 protein expression in normal bile
canalicular membrane vesicles (25), the apparent electrophoretic mo
bility of Mdr2 was distinct from that of Mdrl and Mdr3 expressed in
the same cells (Fig. 1C). We noted a marked difference between the
level of mdr2 mRNA expressed in our cell clones (Fig. IA) and the
final amount of Mdr2 polypeptide produced in these cells (Fig. 1C).
Although the reason of this discrepancy remains unclear, it could
result from (a) a poor translatability in these cells of the mdr2-DHFR
hybrid mRNA produced from intact or rearranged copies of the
transfected plasmid, (b) impaired processing or posttranslational mod
ification of the Mdr2 protein leading to degradation by cellular
proteases, or (c) cellular toxicity of the Mdr2 protein, resulting in
(2—6)
obtainedbythisselectionidentifiedveryabundant
mdr2-DHFR negative selection against high levels of expression.
chimeric transcripts of heterogeneous size (Fig. IA, Lanes 2 and 3).
We next analyzed the functional consequences of Mdr2 protein
These heterogeneous mRNAs probably reflect the use of several
expression on cellular resistance to the MDR-type drugs VBL and
cryptic polyadenylation signals within the 3' ends of the mdr2 and
COL. For this, the drug dose necessary to reduce the plating efficiency
DHFR cDNAs, as well as the SV4O polyadenylation
signal engineered
of mdr2 transfected clones by 50% was established in a cell survival
downstream the expression cassette. The hybridization signal was
assay and was compared to that of control drug-sensitive nontrans
specific, as shown by (a) the lack of hybridization of the mdr2 probe
fected DUK cells; multidrug-resistant mdrl transfectant (EX4N-7)
to control RNAs from multidrug-resistant transfectants expressing
expressing low levels of the Mdrl protein (41), which are comparable
high levels of mdrl or mdr3 (Fig. IA, Lanes 5 and 6), and (b) positive
to those detected in our highest expressing mdr2 transfectants (see
Fig. 3A), and clone 1-2, which expresses high levels of the Mdrl/
hybridization to RNA from a multidrug-resistant transfectant (clone
1-2) expressing a chimeric mdrl/mdr2 gene (16) containing an mdr2
Mdr2 chimeric protein. The results of a typical drug survival assay are
segment overlapping the hybridization probe (Fig. IA, Lane 1). Re
shown for one mdr2 transfectant (clone 2-6), control DUK cells,
EX4N-7 cells, and highly drug-resistant 1-2 cells in Fig. 2; the
hybridization of the Northern blot with mdrl and mdr3 gene-specific
probes confirmed that the mdr mRNA expressed in clone 2-6 was
combined analysis of 6 individual mdr2 transfectant clones is tabu
indeed mdr2 (data not shown). The level of Mdr2 protein expression
lated in Table 1. High level Mdrl-Mdr2 chimeric protein expression
in clone 1-2 resulted in 52- and 38-fold resistance to VBL and COL,
in 10 independent cell clones obtained by this selection protocol was
respectively, while low level Mdrl protein expression in EX4N-7
determined by immunoprecipitation using the monoclonal anti-P-gp
antibody C219 (Fig. 1B). This antibody is directed against the peptide
produced moderate but readily detectable levels of resistance to the
epitope VQE/AALD (45), which is precisely conserved in all three
same drugs (VBL, 6-fold; COL, 4-fold). By contrast, none ofthe mdr2
transfectants showed any detectable degree of VBL or COL resistance
mouse Mdr proteins. This analysis showed variable levels of Mdr2
over the background levels measured in the control drug-sensitive
protein expression in these clones ranging from barely detectable (Fig.
DUK cells. Similar results were also obtained with MDR drugs
1B; Lanes 1, 3, and 4) to intermediate (Fig. 1B; Lanes 5 and 7—10)to
1
2
3
4
5
6
‘i@
I
2
3
4
5
6
7
8
9
10 11 12
I
2
3
456
( 200
: 200
C 100
100
4.5.
A
B
C
Fig 1. Expression of mdr mRNAs and proteins in control and mdr-transfected Chinese hamster ovary cells. (A) Northern analysis of total cellular RNA of tndr-transfccted cell clones.
Ten @sgof total cellular RNA from a cell clone transfected and overexpressing an mdrl/mdr2 chimeric eDNA containing the linker domain of mdr2 inserted into mdrl (clone 1-2,
Lane I); transfected cell clones expressing either mdr2 (clone 2-6; Lanes 2 and 3), mdrl (clone 1-1; Lane 4), or mdr3 (clone 3-5; Lane 5); or control untransfected LR73 cells (Lane
6) wereanalyzedby Northernblottingwitha [32P]-labeled
410-basepairmdr2gene-specific
probeoverlapping
thelinkerdomain(nucleotides
1869—2276).
Ordinate,estimated
size
of hybridizing mRNAs in kilobases (Kb). (B) P-glycoprotein iinmunoprecipitation in independent mdr2-transfected cell clones. [35S]Methionine metabolically labeled total cellular
proteins from 10 independent mdr2-expressing clones (Lanes 1—10),
the drug-resistant clone 1-2 expressing an MdrlfMdr2 chimeric protein (Lane I I), and drug-sensitive DUK cells
(Lane 12) were immunoprecipitated with the anti-P-glycoprotein monoclonal antibody C219 (dilution 1:100) and separated by SDS-PAGE on a 7.5% gel. Molecular mass standards
in kilodaltons were myosin (200 kilodaltons) and phosphorylase B (97 kilodaltons). The gel was fixed, dried, and exposed to X-ray film for 2 weeks (C). Immunoprecipitation of
P-glycoproteins encoded by mdri, mdr2, and mdr3. Immunoprecipitation methods and cell clones were as described in A. Lane 1, LR73 control cells, Lane 2, clone 1-1 (nsdri), Lanes
3 and 5, DUK cells, Lane 4, clone 2-6 (mdr2) and Lane 6, clone 3-5 (mdr3). The gel was fixed, dried, and exposed for 3 days.
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DRUG BINDING TO Mdr2
The M,., 90,000—100,000 C219-reactive peptide present in these mem
brane fractions represents a degradation product of the intact protein
that has been described previously (34). Photolabeling studies of
Mdrl and Mdr2 with [32P]8-azido-ATP showed that both proteins
could be labeled to comparable levels by this photoaffinity probe,
suggesting that they can bind Al? with a similar affinity (Fig. 3C;
mdrl clone EX4N-7, Lane 1; mdr2 clone 2-6, Lane 2). In this
experiment, a membrane-enriched
fraction from an independent
mdr2-transfected cell clone (clone 2-G7; Fig. 3C, Lane 3) also bound
Al? at an equivalent level to Mdrl expressed in EX4N-7 (Fig. 3C,
Lane 1). These results are in agreement with our previous observa
tions that both nucleotide-binding folds of Mdr2 are functional and
can complement the biological activity of Mdrl in chimeric molecules
constructed between the two proteins (16). These results also indicate
that the Mdr2 protein is properly targeted to the membrane fraction of
transfected cells and that it is folded in an appropriate configuration
which allows Al? binding. However, these experiments do not cx
dude the possibility that the Al?ase activity of Mdr2 may be quan
titatively or qualitatively different from that of Mdrl. On the other
hand, photolabeling studies on serial dilutions of membrane fractions
from Mdrl and Mdr2-expressing cells with the drug analogue IAAP
produced strikingly different results. IAAP labeling of Mdr2 protein
was much less pronounced than labeling of Mdrl for an equal amount
of protein loaded (Fig. 3B). These differences in IAAP labeling of
Mdrl and Mdr2 proteins were consistently detected in independent
experiments. Taken together, these results show that although Mdrl
and Mdr2 can combine an Al? analogue with similar efficiency,
binding of a drug analogue to Mdr2 is greatly reduced, suggesting that
reduced drug binding may underlie the incapacity of Mdr2 to transport
these types of substrates.
VINBLASTINE
---0--j
DUK
112
2.6
EX4N7
U
a
U
E
a
a
4@
a
10
100
1000
Vinblastine (ng/ml)
COLCHICINE
---0--S
DUK
112
—P1-—
2-6
—.-—0--
EX4N7
U
5
U
E
as
a
a
DISCUSSION
a
10
100
1000
10000
Colchicine (ng/ml)
Fig 2. Drug survival characteristics of cell clones expressing Mdrl or Mdr2 proteins.
A total of 5000 cells from mdr2-transfected clone 2-6, the mdrl-transfected clone EX4N-7
(both expressing equivalent amounts of protein), the highly multidrug-resistant clone 1-2
(112) expressing the chimeric Mdrl-Mdr2 protein, and control DUK cells (DUK) were
plated in medium containing increasing concentrations of VBL and COL and grown for
3 days. Plating efficiency represents the percentage of cells surviving at a given drug
concentration compared with the same cells grown in medium without drug.
actinomycin
@
D, ADM,
and gramicidin
D (data
not shown).
Several studies have established that as opposed to the mouse mdrl
and mdr3 and human MDRJ genes, transfection of full-length cDNAs
for either mouse mdr2 or its human counterpart MDR2 into otherwise
drug-sensitive cells fails to yield multidrug-resistant clones on plating
the transfected cells into drug-containing medium (1 1, 16, 17). In the
case of human MDR2, stable expression of the protein in transfected
BRO cells fails to increase the resistance of these cells to MDR-type
drugs (17). The goals of the present study were (a) to determine the
phenotypic consequences on drug resistance of stable mouse Mdr2
protein expression in transfected cells, and (b) to investigate the
biochemical basis for the apparent lack of biological activity of Mdr2
These
Table 1 Drug survival characteristics of control and independent mdr transfected
results indicate that as opposed to low level expression of Mdrl (41)
(EX4N-7, Fig. 2) or Mdr3 (28), low level Mdr2 protein expression
fails to confer multidrug resistance in transfected CHO cells. These
observations are in agreement with our previous inability to di
rectly select multidrug-resistant
mdr2 transfectants
in drug
containing medium.
We next wished to investigate the biochemical basis for the func
tional differences detected between Mdrl and Mdr2 proteins in trans
fected cell clones. In particular, we compared the capacity of Mdrl
and Mdr2 to bind the photoactivatable drug analogue IAAP and the
photoactivatable Al? analogue 8-azido-ATP. For this, we used mem
brane-enriched fractions prepared from one of the Mdr2-expressing
clones (2—6)and from the Mdrl-expressing clone EX4N-7. Western
blotting analysis of serial dilutions of these membrane fractions with
the monoclonal anti-P-gp antibody C219 confirmed that both cell
clones produced similar amounts of the respective proteins (Fig. 3A).
Chinese hamster ovary cell clones, expressing either Mdrl or Mdr2 proteins
The various cell clones analyzed are described in the text.
VBL (ng/ml)
COL (ng/ml)
DUK
EX4N-7
1/2
Cell clone
3.9°
25 (6x)―
210(53X)
70
(4X)
2700 (38X)
2-3
2-6
2-8
3.1 (0.8X)
5.3(1.3X)
2.9 (0.7X)
2-64
3.1 (0.8X)
25 (0.4X)
2-68
2-G7
2.9 (0.7x)
6.0 (1.5X)
30 (0.4x)
50 (0.7X)
35 (0.5X)
70(1X)
35 (0.5 X)
a Drug concentrationrequired to reduce plating efficiency of control and mdr-trans
fected cell clones by 50%, as determined in a 3-day cytotoxicity assay. Measurements are
the means of two experiments carried out in duplicate.
b The degree of drug resistance of individual mdr-transfected
cell clones is expressed
as a fold resistance (in parentheses) and was obtained by dividing the drug concentration
required to reduce plating efficiency by 50% value measured for each clone by the drug
concentration required to reduce plating efficiency by 50% of the control drug-sensitive
parental cells.
4895
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1994 American Association for Cancer Research.
DRUG BINDING TO Mdr2
Mdr.Z
12.5
I
25
MdrJ.
50
12.5
25
I
C
50
2
3
4
5
6
50
II
(200
200
j'g
iii
@2O0
I
100
60
100
..‘100
A
B
C
Fig 3. Ligand-binding characteristics of Mdrl and Mdr2 proteins expressed in membrane fractions from transfected cell clones. (A) Western blot analysis of membrane fractions
from cell clones expressing equivalent amounts of Mdrl and Mdr2 proteins. Purified membrane fractions (12.5, 25, and 50 @g)isolated from mdr2-transfected clone 2-6 (Mdr2),
mdrl-transfected clone EX4N-7 (Mdrl), and untransfected LR73 control cells (C) were analyzed by Western blotting. The immunoblot was incubated with the anti-P-gp antibody C219
(1:300 dilution), and specific antigen-antibody complexes were visualized using a goat anti-mouse IgG antisera coupled to alkaline phosphatase. Ordinate, positions of molecular mass
markers in kilodaltons. (B) Photoaffinity labeling of Mdrl and Mdr2 isoforms with IAAP. Purified membrane fractions from the mdrl-transfected clone EX4N-7 (Lanes 1—3)
and the
mdr2-transfected clone 2-6 (Lanes 4—6)were incubated with IAAP (30 nti, final concentration) and cross-linked with UV. The amounts of purified photolabeled membrane proteins
loaded onto the gel were 40 (Lanes 1 and 4), 80 (Lanes 2 and 5) and 160 (Lanes 3 and 6) @.tg.
Mdr proteins were immunoprecipitated using the mouse monoclonal anti-P-gp antibody
C219. Reaction products were separated by SDS-PAGE and the gels were dried and exposed to X-ray film for 1 week. No specific IAAP-labeled product was detected by C219 in
160 @.tg
of purified membranes from control LR73 cells (not shown). Right ordinate, positions of molecular mass standards in kilodaltons. (C) Photoaffmity labeling of Mdrl and Mdr2
isoforms with [a-32P]8-azidoadenosine-5'-triphosphate
(Al?). Purified membrane fractions (100 @.tg)from the mdrl-transfected cell clone EX4N-7 (Lane I), mdr2-transfected clones
2-G7 (Lane 2) and 2-6 (Lane 3) and control untransfected LR73 cells (Lane 4) were incubated with [a-32P18-azido-ATP and cross-linked with UV. Mdr proteins were
immunoprecipitated with the mouse anti-P-gp monoclonal antibody C219 and reaction products were separated by SDS-PAGE as described in B. The gel was exposed to X-ray film
for 3 weeks. Left arrows, different migration of the two AlT-labeled Mdr isoforms (upper arrow, Mdrl ; lower arrow, Mdr2). The length of the resolving gel in A (5 cm) was different
from that used in gels shown in B and C (15 cm).
toward MDR drugs, in particular to compare the ATP and drug
binding characteristics of Mdr2 to those of the biologically active
Mdrl protein. The isolation of transfectants expressing readily detect
able amounts of Mdr2 protein in cell membrane fractions proved
difficult by standard transfection procedures using a cotransfected
dominant selectable marker such as pSV2neo (data not shown). To
circumvent this difficulty, it was necessary to introduce the full-length
mdr2 cDNA in an expression vector which directs the synthesis of
high levels of a bicistronic mdr2-DHFR mRNA with a viral EMC
translation reinitiation sequence positioned immediately upstream the
DHFR
portion
of the mRNA.
Transfection
of this construct
into
DHFR mutant CHO cells, followed by stepwise selection of trans
fected cells in MTX-containing cultured medium, allowed the isola
tion of several cell clones stably expressing fairly abundant amounts
of the Mdr2 protein. Drug cytotoxicity assays showed that Mdr2
expression in independent cell clones failed to increase their levels of
resistance to MDR drugs such as VBL and COL, while similar levels
of Mdrl protein expression in the same cells readily conferred mul
tidrug resistance as measured in the same assay. Biochemical analysis
of the Mdr2 and Mdrl proteins in cross-linking experiments using
membrane-enriched fractions from transfected cells, photoactivatable
analogues of Al?, and the known P-gp ligand LAAP showed that
although both proteins bound the ATP analogue to the same extent,
binding of I.AAP to Mdr2 was significantly reduced when compared
to Mdrl. These data indicate that as opposed to Mdrl, Mdr2 does not
modulate drug efflux in these transfectants, and that this inability to
confer drug resistance is linked to decreased drug binding to Mdr2. It
is also interesting to note that abolition of ADM and COL resistance
in an mdrl mutant bearing a unique substitution at position 941
(TM1 1) results in approximately 75% loss of IAAP binding to the
protein (46), a reduction similar to the differences detected here
between wild type Mdrl and Mdr2 proteins (Fig. 3B).
Several lines of evidence suggest that our findings on Mdr2 in
transfected CHO cells are an accurate reflection of functional differ
ences between
Mdr2 and Mdrl/Mdr3
proteins,
rather than a particu
targeting, posttranslational protein modification, or folding of an
otherwise biologically active protein. Transfection of mdr2 in several
additional cell types from distinct anatomical origins, including hepa
tocytes derived cell lines (the site of normal mdr2 expression) fails to
yield drug-resistant cell clones. Expression of equivalent amounts of
Mdrl and Mdr2 proteins in the same cell type confer increased drug
resistance only in the Mdrl-expressing cells (Fig. 2, Table 1). Western
blotting analysis of highly enriched membrane fractions from mdr2
transfectants shows abundant Mdr2 protein expression in this fraction,
suggesting appropriate membrane targeting of the protein (Fig. 3A).
The photoactivatable Al? analogue 8-azido-Al? labels to the same
extent Mdr2 and Mdrl both expressed at the same level in these
membrane fractions (Fig. 3C), suggesting appropriate Mdr2 protein
folding in the membrane of these transfected cells. The reduced
binding ofthe drug analogue IAAP to Mdr2 detected in the membrane
fractions of transfected cells (Fig. 3B) is in agreement with the
absence of IAAP binding to Mdr2 expressed in normal liver canalic
ular membrane vesicles reported previously by our group (25).
Our previous studies with chimeric Mdrl/Mdr2 molecules con
structed by exchanging homologous protein domains have indicated
that both NB sites of Mdr2 are functional and can complement the
biological activity of Mdrl in transfection experiments (16). Although
these findings do not establish that the Al?ase activity of Mdr2 is
similar to that of Mdrl, they do indicate that both NB sites are
interchangeable in these proteins. Likewise, both the highly divergent
@2termi@ segment and the linker region of Mdr2 can be substi
tuted in Mdrl without loss of Mdrl biological activity (16).@ How
ever, the substitution of small or large protein segments overlapping
the membrane-associated regions of Mdrl by the homologous do
mains of Mdr2 completely abolished the ability of the resulting
chimeras to confer MDR, suggesting that these regions are function
ally distinct in the two proteins. Similar results have also been
obtained with a chimeric human MDR1 protein bearing a small
TM2-TM3 segment derived from human MDR2 (47). These data,
larity of the in vitro cell expression system used here where the
specific cellular background could lead to inappropriate membrane
4896
4 P. Gros
and
E. Buschman,
unpublished
observations.
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1994 American Association for Cancer Research.
DRUG BINDING TO Mdr2
together with those reported here, suggest that reduced IAAP binding
to Mdr2 compared to Mdrl may be linked to functional differences in
the membrane-associated segments of Mdr2. Indeed, the genetic anal
ysis of mutant P-gps showing altered substrate specificity has shown
that mutations in or near predicted TM domains in both halves of the
proteins affect drug transport by modulating the drug-binding char
acteristics of the protein, including those of IAAP (27—31,46, 48).
Recently, the binding site of IAAP in Mdrl (Mdrlb) has been dc
gantly localized by epitope mapping of proteolytic radiolabeled frag
ments to two small peptides near predicted TM6 (minor site) and
TM12 (major site) of the protein (32, 35, 36). A comparison of the
predicted amino acid sequences of Mdrl and Mdr2 in these regions
reveals very few nonconservative substitutions (jositions 968—969
and 1002—1006). It is tempting to speculate that these amino acid
residues may play a key role in forming a binding pocket for IAAP,
which may be disrupted in Mdr2. The functional role of these amino
acid residues in drug binding can now be addressed by site-directed
mutagenesis and by using the expression system described in this
study.
Finally, our finding that (a) the ability of Mdr2 to bind 8-azido-Al?
is equivalent to that of Mdrl, and that (b) both predicted NB sites of
Mdr2 can functionally complement the homologous NB sites of Mdrl
(16) are in agreement with the proposal that Mdr2 may act on
non-drug substrates. In normal mouse liver, the very abundant and
polarized expression of Mdr2 in the canalicular membrane of epithe
hal cells of the biliary ductules (25), and the specific phenotype of
mouse mutants lacking mdr2, indeed suggest that Mdr2 may transport
PC by a lipid flippase mechanism (26).
ACKNOWLEDGMENTS
The authors thank France Talbot for technical assistance and Dr. R.
Kaufman (Genetics Institute, Cambridge, MA) for the generous gift of plas
mids pEMC2b1
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4898
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The Inability of the Mouse mdr2 Gene to Confer Multidrug
Resistance Is Linked to Reduced Drug Binding to the Protein
Ellen Buschman and Philippe Gros
Cancer Res 1994;54:4892-4898.
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