Tài liệu Báo cáo khoa học: Competition between innate multidrug resistance and intracellular binding of rhodamine dyes pdf

Competition between innate multidrug resistance and
intracellular binding of rhodamine dyes
Daniella Yeheskely-Hayon, Ronit Regev, Hagar Katzir and Gera D. Eytan
Department of Biology, The Technion – Israel Institute of Technology, Haifa, Israel
A major obstacle impeding the success of chemother-
apy is multidrug resistance (MDR). MDR in patients
is exhibited as a resistance to a wide variety of struc-
turally unrelated drugs. This is caused by several
factors, one of which is ‘classical’ MDR characterized
by diminished cellular uptake of the drugs due to
active export by one or more ABC transporters. The
ABC transporter most often over-expressed in vitro in
cells exposed to increasing drug concentrations is
plasma glycoprotein (Pgp, ABCB1) [1–3]. This protein
is an active efflux pump for chemotherapeutic drugs,
natural products and hydrophobic partially positive
dyes. The ABC transporter superfamily is represented
in humans by 48 members [4,5], of which 24 are
known to function as drug transporters relevant to
various diseases. In addition to Pgp, the multidrug
resistance-associated protein (MRP1, ABCC1) [6] and
breast cancer resistance protein (BCRP⁄ MXR ⁄ ABCP,
Keywords
innate multidrug resistance; MDR; MRP1;
multidrug resistance; rhodamines
Correspondence
G. D. Eytan, Department of Biology, The
Technion – Israel Institute of Technology,
Haifa, Israel
Fax: +972 4 8225153
Tel: +972 4 8293406

E-mail:
Website:
(Received 7 September 2008, revised 12
November 2008, accepted 20 November
2008)
doi:10.1111/j.1742-4658.2008.06812.x
The present study aimed to elucidate the contribution of the intracellular
binding of drugs to multidrug resistance. For this purpose, uptake of rhod-
amines was studied in cells whose mitochondria had been uncoupled with
carbonyl cyanide m-chlorophenylhydrazone. Surprisingly, in a variety of
drug-untreated cells, presumed to be sensitive to multidrug resistance-type
drugs, rhodamines were excluded from entering the cells. Thus, the amount
of rhodamine 123 taken up into parental untreated K562 cells was less than
the amount bound to the cell exterior. Rhodamine uptake was prevented
by an active efflux pump. The efflux was inhibited by 4-chloro-7-nitro-
2,1,3-benzoxadiazole (NBD-Cl) and MK571 and, to a lesser extent, by
ATP depletion, indomethacin, probenecid and vanadate. All the inhibitors,
apart from NBD-Cl, are known to modulate multidrug resistance-associ-
ated protein (MRP) 1. Because MRP1 was expressed in all the cell lines
tested and the efflux of rhodamines in MRP1 over-expressing cells was
abolished by NBD-Cl, it appears that rhodamines are excluded from these
cells by MRP1. On the other hand, the uptake of rhodamines into cells
respiring with their coupled mitochondria demonstrated diminished sensi-
tivity to NBD-Cl and MK571. Thus, active pumping into the mitochondria
allowed enhanced uptake into the cells, overcoming the innate resistance.
The innate resistance provided by MRP1 to cells prevents rhodamine dyes,
and possibly drugs such as doxorubicin, from achieving equilibration of
their concentration in the cytoplasm with their concentration in the exter-
nal medium. The protection provided to multidrug resistance cells by ABC
transporters has to overcome competition by passive uptake of the drugs

and binding ⁄ uptake of the drugs into intracellular targets.
Abbreviations
CCCP, carbonyl cyanide m-chlorophenylhydrazone; MDR, multidrug resistance; MRP, multidrug resistance-associated protein; NBD-Cl,
4-chloro-7-nitro-2,1,3-benzoxadiazole; Pgp, plasma glycoprotein; TMR, tetramethylrhodamine; TMRM, tetramethylrhodamine methyl ester.
FEBS Journal 276 (2009) 637–648 ª 2008 The Authors Journal compilation ª 2008 FEBS 637
ABCG2) [7–9] have been well-established as efflux
pumps preventing the penetration of anticancer-drugs
into tumor cells in vitro and in patients.
For MDR research, highly resistant cells have been
generated by the exposure of sensitive parent cell lines
to increasing concentrations of an anticancer drug.
The resulting resistant cells expressed high levels of
ABC transporters compared to the minute expression
of these proteins in parent lines. It has been assumed
that the low levels of ABC transporters present in the
parent cell lines cannot cope with significant drug con-
centrations and, as a result, drug uptake into sensitive
cells is not affected by pump activity but is due to pas-
sive transmembrane transport [10–12]. In addition,
upon exposure of cells to drugs, a steady-state is
achieved, with the free drug concentration in the cyto-
plasm being equivalent to its concentration in the
extracellular medium [10,13,14].
Pgp is a key player in the defense of the body
against amphipathic xenotoxins. At the blood–brain
barrier, and in placental trophoblasts, testis and bone
marrow, it provides protection of vital body parts and,
in the gut, liver and kidney, Pgp helps to eliminate tox-
ins from the body [15–18]. On the other hand, MRP1
is present in virtually all human tissues and in most

human tumor cell lines and tumor samples [4]. Using
mice genetically deficient in both the mdr1a and mdr1b
genes [mdr1a ⁄ 1b() ⁄ )) mice], the mrp1 gene [mrp1() ⁄ ))
mice] and the combined genes mdr1a ⁄ 1b and mrp1
[mdr1a ⁄ 1b() ⁄ )), mrp1() ⁄ )) mice], it has been shown
that the innate levels of these ABC transporters,
expressed in mice, confer MDR [19–21]. BCRP is
expressed specifically in human placenta, liver and
breast [22].
The evidence presented below indicates that a
variety of ‘sensitive’ human cell lines exhibit MDR
activity capable of preventing the accumulation of
rhodamine dyes in the cell cytoplasm. These cationic,
relatively hydrophobic dyes, especially rhodamine 123,
have been widely used as probes for MDR ex vivo.
Inside the cells, these dyes are actively driven into the
mitochondria by the respiratory electrochemical poten-
tial [23]. In the presence of uncouplers that abolish
this active accumulation, such as carbonyl cyanide
m-chlorophenylhydrazone (CCCP), rhodamine dyes
entering the cells remain free in the cytoplasm and are
passively bound to various intracellular sites. Likely
sites are the intracellular membranes because they are
hydrophobic and negatively charged. A comparison of
rhodamine uptake, in the presence and the absence
of uncouplers, allows the estimation of the effect of
intracellular binding on the MDR phenomenon. The
data detailed obtained in the present study indicate
that innate cellular MDR interferes successfully with
the accumulation of dyes, and probably certain drugs,

in the cytoplasm. On the other hand, high-affinity
binding of these agents to intracellular receptors over-
whelms the low levels of ABC transporters present in
‘sensitive’ cells, and higher expression levels of ABC
transporters are required to prevent binding to these
receptors.
Results
The amount of rhodamine dyes associated with K562
cells was determined. The cells were separated from
the external medium by centrifugation through an oil
cushion and dissolved prior to determination of the
amount of dyes associated with them. The advantages
of such an assay are that: (a) it is quantitative; (b) it is
not affected by intracellular quenching of the dyes;
and (c) it measures the total dye amount associated
with the cells, including the dye adsorbed to the sur-
face of the cells.
To avoid complication of the uptake studies by
active pumping of the dyes into the mitochondria, dye
uptake was measured routinely in the presence of the
mitochondrial uncoupler, CCCP, The quantities of
rhodamine 123, tetramethylrhodamine (TMR), tetram-
ethylrhodamine methyl ester (TMRM) and rhodamine
6G adsorbed to the cell surface under these conditions
were equivalent to the rhodamine amounts dissolved in
medium volumes equal to 1.3, 9.1, 2.3 and 4.8 cell vol-
umes, respectively (Fig. 1 and Table 1). TMR exhib-
ited the highest affinity toward the cell plasma
membrane, whereas rhodamine 123 exhibited the low-
est affinity. Within the limitations imposed by rhoda-

mine dye solubility in the medium, the apparent
volume of dye adsorbed to the cells was independent
of the dye concentrations used (Fig. 2). Thus, it
appears that the rhodamine dyes are bound to sites
abundant on the cell surface, rather than to specific
receptors. As expected, the omission of mitochondrial
poisons did not affect the amount of dye adsorbed to
the cell surface.
Incubation of cells at room temperature with rhoda-
mine dyes resulted in dye uptake into the cells that
reached apparent quasi-equilibrium within < 1 h. In
agreement with an earlier study [14], the cellular
uptake of rhodamine 123 was slow and the cells
reached quasi-equilibrium within 1 h when incubated
at 37 °C. Under conditions designed to eliminate the
active uptake of the dyes into mitochondria by poison-
ing the latter with CCCP, azide or cyanide, the
amount of rhodamine dye accumulated within the cells
was low (Fig. 1). The amount of rhodamine 123 taken
Innate multidrug resistance to rhodamines D. Yeheskely-Hayon et al.
638 FEBS Journal 276 (2009) 637–648 ª 2008 The Authors Journal compilation ª 2008 FEBS
up into the cells was less than the amount adsorbed to
the cell surface. The amounts of the other rhodamine
dyes were somewhat higher, but still < 3.5 times the
amounts adsorbed to the cell surface.
Because the amount of intracellular membranes
exceeds that of the plasma membrane, it was expected
that, under quasi-equilibrium conditions, the amount
of rhodamine dye located within the cells would be
much higher compared to the amount adsorbed to the

cell surface. Indeed, rendering cells permeable with dig-
itonin allowed the enhanced uptake and binding of all
the rhodamines (data not shown). To explore the pos-
sibility that the rhodamine dyes were prevented from
penetrating into cells by an ATP-dependent pump
mechanism, we depleted the ATP content of cells by
depriving them of glucose in addition to poisoning
their mitochondria. Under these conditions, cellular
ATP levels were reduced ten-fold (as determined by
15
200
5
10
100
Rhodamine 123 TMR
+ CCCP
– CCCP
0 15304560
0
0102030
0
On ice + CCCP
40
60
40
60
Rhodamine 6GTMRM
0102030
0
20

0102030
0
20
Apparent concentrations ratio (cell/medium)
Time (min)
Fig. 1. Uptake kinetics of rhodamine ana-
logues into K562 cells. K562 cells were
incubated either on ice, at 37 °C (rhodamine
123) or at 23 °C (other rhodamine dyes), for
15 min in the absence or presence of CCCP
(1 l
M). Subsequently, 25 lM of rhodamine
123, TMR, TMRM or rhodamine 6G was
added and the cells were incubated further.
At various time points, cell samples were
withdrawn and the amount of dye associ-
ated with the cells was determined after
their separation from the external medium
by centrifugation through an oil cushion as
described in the Experimental procedures.
The apparent concentrations ratio was
calculated assuming a cell volume of
2.5 l
3
[49]. Data represent the mean ± SD
of four independent experiments.
Table 1. Uptake kinetics of the various rhodamine analogues into K562 cells. The raw experimental results are presented in Fig. 1. The
inhibitor used was either NBD-Cl or MK571. The dye amount adsorbed to the cell surface was estimated as the dye quantity associated with
cells incubated on ice at the first time point. This amount was subtracted from the various curves to obtain the amount of dye taken up into
cells. The uptake data were fitted using the equation: dye uptake = c · (1 – e

)kt
). The amount of dye taken up by the cells at quasi-equilib-
rium conditions is equal to c. The half time of the uptake curve was calculated using the constant, k. The amount of dye associated with the
cells is expressed as the apparent cell volume of dye bound to a cell, which is equivalent to the concentration ratio of dye bound to the cells
versus extracellular dye. The quality of the fitting is expressed in terms of R
2
.
Uncoupler
Uncoupler
+ inhibitor Control
Control
+ inhibitor
Rhodamine 123 Half time Minutes 21.7 14.5 14.1 14.7
Dye uptake Cell volumes 1.2 10.5 9.3 8.9
R
2
0.93 0.96 0.97 0.95
TMR Half time Minutes 2.6 6.8 6.6 4.8
Dye uptake Cell volumes 31.6 193.0 134.1 255.6
R
2
0.83 1.00 0.99 0.98
TMRM Half time Minutes 2.6 6.9 10.2 7.3
Dye uptake Cell volumes 5.6 51.7 48.7 56.7
R
2
0.77 1.00 1.00 0.98
Rhodamine 6G Half time Minutes 6.3 4.2 6.7 5.8
Dye uptake Cell volumes 13.9 60.8 44.2 64.0
R

2
0.98 0.96 0.98 0.98
D. Yeheskely-Hayon et al. Innate multidrug resistance to rhodamines
FEBS Journal 276 (2009) 637–648 ª 2008 The Authors Journal compilation ª 2008 FEBS 639
the luciferin–luciferase assay and confirmed in a previ-
ous study [24]) and this resulted in a doubling of the
amount of TMRM taken up into the cells. To charac-
terize further the mechanism responsible for the
exclusion of rhodamine dyes from cells, we surveyed a
variety of agents known to interfere with the trans-
port of anticancer drugs and dyes in and out of
cells (Table 2). 4-Chloro-7-nitro-2,1,3-benzoxadiazole
(NBD-Cl) caused a prominent increase in rhodamine
dye uptake into cells (Fig. 3 and Table 2). Further-
more, it had a marked effect in cells whose mitochon-
dria had been poisoned and a relatively small effect in
cells with unpoisoned mitochondria (Fig. 3).
Fluorescence microscopy confirmed that the amount
of rhodamine dye taken up into cells whose mitochon-
dria had been poisoned was very low (Fig. 4). The low
fluorescence of TMRM present in K562 cells was dif-
fuse throughout the cytoplasm and the nucleus. As
expected, omission of the uncoupler allowed a massive
accumulation in the mitochondria. The fluorescence
level and location of TMRM taken up into respiring
cells was not altered by NBD-Cl. On the other hand,
NBD-Cl enhanced the fluorescence of TMRM taken
up into nonrespiring cells, but had little effect on the
distribution of the dye within the cells.
NBD-Cl is known to inactivate Pgp in vitro [25].

However, this inactivation was prevented by the pres-
ence of ATP [25]; thus, the increased uptake of rhoda-
mine dyes observed in the presence of NBD-Cl does
not appear to be due to the inhibition of Pgp. A half
maximal effect of NBD-Cl was observed at concentra-
tions of 3–5 lm, irrespective of the rhodamine concen-
trations that the cells were exposed to, indicating that
the direct effect of the NBD-Cl was on a cellular com-
ponent and not on the rhodamine dye (Fig. 5A).
Moreover, incubation of rhodamine dyes with NBD-
Cl, either in presence of cells or in their absence, did
not affect the fluorescence spectrum of the dyes (data
not shown). To exclude the possibility that the effect
of NBD-Cl is due to inactivation of the uncoupler
activity of CCCP, the data were reproduced with the
60
250
12

– CCCP
20
40
50
100
150
200
3
6
9
TMRMRhodamine 123 TMR

+ CCCP
100 10 1 0
0
100 10 1 0
0
100 10 1 0
0
Apparent concentrations
ratio
(cell/medium)
Dye concentration (µM)
On ice + CCCP
Fig. 2. Effect of rhodamine dye concentrations on their uptake into K562 cells. K562 cells were incubated either on ice for 2 min or for 1 h,
either at 23 °C (TMR and TMRM) or at 37 °C (rhodamine 123), in the presence of various concentrations of TMRM, TMR or rhodamine 123.
The incubation medium contained CCCP in the absence or presence of NBD-Cl (20 l
M). Subsequently, cell samples were withdrawn and
the amount of dye associated with the cells was determined after their separation from the external medium by centrifugation through an
oil cushion as described in the Experimental procedures. Data represent the mean ± SD of four independent experiments.
Table 2. The effect of various agents on TMRM accumulation in
K562 cells. ‘Sensitive’ cells were incubated at 23 °C for 15 min
with the various agents and 1 l
M CCCP. Subsequently, TMRM
(10 l
M) was added and the cells were incubated further for 30 min
at room temperature. The amount of TMRM associated with the
cells was measured as described Fig. 1. The amount of dye
adsorbed to the cell surface was estimated as described in Fig. 1.
Increase in
TMRM
uptake (%)

ATP depletion Preincubation
for 60 min with
10 m
M
deoxyglucose
instead of
glucose
104 ± 17
NBD-Cl 20 l
M 470 ± 83
MK571 50 l
M 420 ± 75
Indomethacin 0.5 m
M 130 ± 42
Probenecid 3 m
M 35 ± 10
Cyclosporine A 10 l
M 0
Fumitremorgin C 5 l
M 0
Vanadate (ortho) 10 m
M 120 ± 18
n-Ethylmaleimide 0.1 m
M 0
N,N¢-Dicyclohexylcarbodiimide 1 m
M 0
p-Chloromercuriphenylsulfonic
acid
1m
M 0

Concanamycin A 20 n
M 0
Glutathione depletion Growth for 48 h
in presence of
50 l
MDL-buthionine-
(S,R)-sulfoximine [48]
0
Innate multidrug resistance to rhodamines D. Yeheskely-Hayon et al.
640 FEBS Journal 276 (2009) 637–648 ª 2008 The Authors Journal compilation ª 2008 FEBS
mitochondrial poisons, KCN (1 mm) and sodium azide
(10 mm), with identical results being achieved.
Similar effects of NBD-Cl on TMRM uptake into
cells were observed with GLC4 (human small cell lung
cancer cell), HEK293 (human embryonic kidney cell),
2008 (human ovarian carcinoma cell), and CEM-
CCRF, CIR and Jurkat (human transformed T cell)
cell lines (Fig. 5B), indicating that this phenomenon is
not specific to K562 cells, but is evident in a variety of
cells.
A reversal of the innate resistance to rhodamine
uptake similar to that obtained with NBD-Cl was
observed with MK571 (Table 2 and Fig. 5). MK571,
an established inhibitor of several MRP transporters
[26–28], allowed the increased uptake of rhodamines
into the multidrug-sensitive K562 cells with a half
maximal effect being observed at 22 lm. The effect of
MK571 on all the cell lines mentioned above was simi-
lar to that of NBD-Cl (data not shown). Probenecid,
another inhibitor of MRP transporters [29], increased

rhodamine uptake (Table 2). Vanadate, a nonspecific
inhibitor of ATPase activity, enhanced TMRM uptake
into the cells. On the other hand, the Pgp chemosensi-
tizer, cyclosporin A, had no effect on rhodamine
uptake. Similarly, n-ethylmaleimide, N,N¢-dicyclohexyl-
carbodiimide and p-chloromercuriphenylsulfonic acid,
and the specific inhibitor of BCRP, fumitremorgin C
[30], as well as glutathione depletion, had no effect on
15
300
5
10
100
200
Rhodamine 123
TMR
+inhibitor + CCCP
+inhibitor – CCCP
0
0
+ CCCP
40
60
40
60
Rhodamine 6G
TMRM
0102030
0153045
60

0102030
0102030
0
20
0
20
Apparent concentrations ratio
(cell/medium)
Time (min)
Fig. 3. Effect of NBD-Cl or MK571 on uptake
kinetics of rhodamine analogues into K562
cells. K562 cells were incubated either at
37 °C (rhodamine 123) or at 23 °C (other
rhodamine dyes) for 15 min in the absence
or presence of CCCP (1 l
M), with either no
addition or with the addition of either 50 l
M
MK571 (rhodamine 123 panel) or 20 lM
NBD-Cl (other panels). Subsequently, 25 lM
of rhodamine 123, TMR, TMRM or rhoda-
mine 6G was added and the cells were
incubated further. At various time points, cell
samples were withdrawn and the amount of
dye associated with the cells was deter-
mined after their separation from the exter-
nal medium by centrifugation through an oil
cushion as described in the Experimental
procedures. The apparent concentrations
ratio was calculated assuming a cell volume

of 2.5 l
3
[49]. Data represent the mean ± SD
of four independent experiments.
Fig. 4. The effect of MK571 and NBD-Cl on TMRM distribution in K562 and 2008 cell lines. K562 cells and 2008 cells were incubated in the
absence or presence of either 50 l
M MK571 or 20 lM NBD-Cl for 15 min at room temperature. TMRM (0.1 lM) was added and the cells
were incubated further in the presence or the absence of 10 l
M CCCP for 30 min. The cells were photographed as described in the Experi-
mental procedures.
D. Yeheskely-Hayon et al. Innate multidrug resistance to rhodamines
FEBS Journal 276 (2009) 637–648 ª 2008 The Authors Journal compilation ª 2008 FEBS 641
the uptake of this rhodamine analogue. As expected
for the active efflux mechanism of rhodamine dyes,
agents allowing the enhanced uptake of rhodamine
dyes inhibited the efflux of these dyes from the cells.
The efflux of rhodamine 123 and TMRM was inhib-
ited by NBD-Cl and MK571 and, to a lesser extent,
by ATP depletion (Fig. 6).
An alternative experimental approach to demon-
strate the uptake of rhodamine dyes into cells is to
follow their quenching upon entering the cells. In
respiring cells, the active concentration of these dyes in
the mitochondria results in quenching of their fluores-
cence. On the other hand, it has been reported that,
when the active uptake of rhodamine dyes into the
mitochondria is disrupted, incubation of cells with
such dyes does not result in quenching of their fluores-
cence [14] (Fig. 7). The data obtained in the present
study suggest that the reason for the observed lack of

fluorescence quenching could be due to prevention of
the uptake of dyes by their active efflux from the cells.
Indeed, inhibition of this efflux mechanism by MK571
or NBD-Cl resulted in fluorescence quenching of
0.15
0.10
0.05
0.00
4
6
2
100
75
50
25 0
0
50 25 0
NBD-C1 (µM)
Cell associated dye
A B
Fig. 5. NBD concentrations required to inhibit TMRM efflux from various cell lines. (A) 10
6
per mL K562 cells were incubated for 15 min at
23 °C in the presence of various NBD-Cl concentrations. Subsequently, either 2 l
M (circles) or 25 lM (diamonds) TMRM was added and the
cells were incubated further for 1 h. The amount of TMRM associated with the cells was determined as described in Fig. 1. The results are
expressed as the fraction of the dye that became cell-associated. (B) Six cell lines were treated similarly, except that the TMRM concentration
was 10 l
M. Circles, GLC4; diamonds, HEK293; squares, 2008; triangles, CEM-CCRF; inverted triangles, CIR; cross haired circles, Jurkat. The
results are expressed as the dye amount associated with the cells relative to the dye amount associated with cells in absence of NBD-Cl.

1.00
0.75
0.50
0.25
0.00
TMRM Rhodamine 123
Relative TMRM content
30 20 10
0
60 45 30 15 0
Time (min)
AB
Fig. 6. NBD-Cl and MK571 inhibit efflux of rhodamine dyes from ‘sensitive’ K562 cells. (A) K562 cells were loaded with TMRM with the
addition of CCCP (1 l
M) by incubation at 23 °C for 30 min in the presence of either 25 lM dye (circles), 5 lM dye and 20 lM NBD-Cl (trian-
gles) or 5 l
M dye and 50 lM MK571 (squares). Another cell sample was depleted of ATP by incubation in a medium containing CCCP and
10 m
M deoxyglucose and in the absence of glucose for 1 h (diamonds). TMRM (10 lM) was added to this sample and the cells were incu-
bated further for 30 min. The various dye concentrations presented to the cells during the loading phase were chosen in order to yield simi-
lar final dye contents despite various loading conditions. Subsequently, all cell samples were pelleted and resuspended in dye-free media
with the same additions as present during the loading of the cells. At various time points, cell samples were withdrawn and the amount of
dye associated with the cells was determined after their separation from the external medium by centrifugation through an oil cushion as
described in the Experimental procedures. (B) K562 cells were incubated for 1 h at 37 °C in the presence of 1 l
M CCCP and 100 lM rhoda-
mine 123. The cells were pelleted and resuspended in dye-free media in the absence (circles) or presence of either 20 l
M NBD-Cl (triangles)
or 50 l
M MK571 (squares). At various time points, cell samples were withdrawn and processed as described above. Data represent the
mean ± SD of four independent experiments.

Innate multidrug resistance to rhodamines D. Yeheskely-Hayon et al.
642 FEBS Journal 276 (2009) 637–648 ª 2008 The Authors Journal compilation ª 2008 FEBS
TMRM (Fig. 7). Similarly, but to a lesser extent, indo-
methacin, probenecid and vanadate allowed the
enhanced uptake of TMRM, resulting in quenching of
its fluorescence. Other agents, such as concana-
mycin A, which had no effect on rhodamine efflux as
assayed by the quantitative assay reported in Table 2,
did not allow quenching of TMRM fluorescence. This
phenomenon has been observed in various cell lines,
including K562, CCRF-CEM, HL-60, T2 and Chinese
hamster ovary cells (Fig. 7).
To explore the possibility that the active efflux of
the rhodamines from cells presumed to be multidrug
sensitive is mediated by one or more of the commonly
studied ABC transporters, we studied the effect of var-
ious efflux inhibitors on TMRM efflux from cell lines
over-expressing Pgp, MRP1, MRP2, MRP3, MRP4
and MRP5 (Fig. 8). Under experimental conditions
optimized to observe the NBD-sensitive efflux of rhod-
amines, the effect of the inhibitors on the cell lines
over-expressing the various transporters was similar to
that observed in their corresponding parent cell lines,
except for the cells over-expressing Pgp. Over-expres-
sion of Pgp reduced the amount of TMRM taken up
into cells in the absence of inhibitors compared to its
parent line. However, this reduction was reversed by
cyclosporin A and, thus, the NBD-Cl-sensitive export
observed in multidrug ‘sensitive’ cells does not appear
to be due to Pgp activity. Because the over-expression

of MRP 1–5 did not significantly reduce the amount
of TMRM taken up into the cells, the obstacle to
uptake of rhodamines into the cells can be due to one
of two possibilities: (a) NBD-Cl-sensitive export is not
mediated by either of these MRPs or (b) the low
amount of MRPs present in ‘sensitive’ cells is sufficient
to prevent the uptake of rhodamine dyes into the cyto-
plasm. In the latter case, the over-expression of the
transporter has no effect, but inhibition of the trans-
porter present in the ‘sensitive’ cells is expected to
reverse the obstacle to rhodamine uptake. Because
MRP-1 is inhibited by MK571 and is an MRP that is
relatively abundant in untreated ‘sensitive’ cells [4], it
is a likely candidate for mediating innate resistance in
these cells. Indeed, as shown in Fig. 9, NBD-Cl inhi-
bited MRP1-mediated efflux, but not efflux mediated
by Pgp.
Discussion
Upon exposure of cells whose mitochondria had been
poisoned to rhodamines, quasi-equilibrium is achieved
within 1 h. Contrary to our expectations, under these
conditions, the free concentration of the dyes in the
cytoplasm is low compared to the extracellular concen-
trations of the dyes. Equilibration of the dyes across
the plasma membrane appears to be prevented by an
active export mechanism, which is capable of handling
relatively high extracellular concentrations of dyes.
This is most evident with rhodamine 123, whose intra-
cellular amount at quasi-equilibrium is less than the
dye amount bound to the cell surface. It is possible

that the small amount of rhodamine 123 taken up into
the cells is located at the inner leaflet of the plasma
Time (min)
Fig. 7. TMRM uptake measured as quenching of the dye fluorescence. Left: CCRF-CEM cells were incubated in fluorimeter cuvettes with
stirring for 15 min at 37 °C in the presence of 10 m
M sodium azide and in the absence or presence of 20 lM NBD-Cl, 50 lM MK571, 2 mM
indomethacin, 2 mM probenecid, 10 mM vanadate or 20 nM concanamycin A. At the time points indicated by arrows, 5 lM TMRM was
added. The TMRM fluorescence was monitored continuously with an excitation wavelength of 555 nm and an emission wavelength of
575 nm. Inhibition of the NBD-Cl-sensitive efflux allowed the uptake of TMRM and its quenching within the cells. Right: the various cell lines
indicated in the figure were incubated for 15 min with the addition of 10 m
M sodium azide and in the absence (upper trace) or presence
(lower trace) of 20 l
M NBD-Cl. Subsequently, TMRM was added and its fluorescence was monitored continuously. CHO, Chinese hamster
ovary.
D. Yeheskely-Hayon et al. Innate multidrug resistance to rhodamines
FEBS Journal 276 (2009) 637–648 ª 2008 The Authors Journal compilation ª 2008 FEBS 643
K562
K562/ADR
GLC4
GLC4/ADR
0.3
0.2
0.1
0.0
0.3
0.2
0.1
0.0
0.3
0.2

0.1
0.0
0.3
0.2
0.1
0.0
2008
MRP1
MRP4
MRP5
MRP2
MRP3
HEK293
Cell associated TMRM (fraction of total)
Control
NBD-Cl
MK571
Indomethacin
Probenecid
Cyclosporin A
Control
NBD-Cl
MK571
Indomethacin
Probenecid
Cyclosporin A
Fig. 8. NBD-Cl-sensitive TMRM uptake into various MDR cells and their wild-type cell lines. Cells were incubated for 15 min at 23 °Cina
medium containing CCCP (1 l
M) in the absence (control) or presence of 20 lM NBD-Cl, 50 lM MK571, 2 mM indomethacin, 2 mM probene-
cid or 5 l

M cyclosporin A. Subsequently, 10 lM TMRM was added and the cells were incubated further for 30 min. The amount of TMRM
associated with the cells was determined as described in Fig. 1. The cells included the K562 cell line and its Pgp over-expressing subline
(K562 ⁄ ADR), the GLC4 cell line and its MRP1 over-expressing subline (GLC4 ⁄ ADR), the 2008 cell line and its sublines over-expressing
MRP1, MRP2 or MRP3, and the cell line HEK293 and its sublines over-expressing either MRP4 or MRP5.
1.00
K562
K562 + NBD-Cl
K562/ADR + NBD-Cl
K562/ADR
0.75
GLC4
GLC4/ADR
GLC4 + NBD-Cl
GLC4/ADR + NBD-Cl
0.25
0.50
3020100
Relative TMRM content
604530150
0.00
Time (min)
Fig. 9. NBD-Cl inhibits MRP1-mediated efflux of TMRM. Cells were incubated for 15 min at 23 °C in CCCP (1 lM) medium in the absence
or presence of 20 l
M NBD-Cl. Subsequently, 5 lM (to control cells) or 1.25 lM (to NBD-treated cells) TMRM was added and the cells were
incubated further for 30 min. The dye concentrations presented to the cells during the loading phase were chosen in order to yield similar
final dye contents despite different loading conditions. The cells were pelleted and resuspended to a concentration of 10
6
cellsÆmL
)1
in dye-

free medium containing CCCP. At the time points denoted, samples were withdrawn and the amount of TMRM associated with the cells
was determined. The cells included the K562 cell line and its Pgp over-expressing subline (K562 ⁄ ADR), and the GLC4 cell line and its MRP1
over-expressing subline (GLC4 ⁄ ADR).
Innate multidrug resistance to rhodamines D. Yeheskely-Hayon et al.
644 FEBS Journal 276 (2009) 637–648 ª 2008 The Authors Journal compilation ª 2008 FEBS
membrane and does not accumulate within the cyto-
plasm at all. A logical assumption is that the affinity
of rhodamines to intracellular membranes is not very
different from their affinity towards the extracellular
surface of the plasma membrane. Because the surface
area of intracellular membranes far exceeds that of the
plasma membrane, it is expected that, upon true equili-
bration of dyes or drugs throughout the cells, their
intracellular amount will be large compared to the
amount bound to the cell surface. Active uptake of
dyes into the mitochondria allows a significant increase
in uptake of rhodamine 123 into MDR ‘sensitive’ cells.
Presumably, the active uptake of the rhodamines into
the mitochondria competes with and overcomes the
efflux pump. Thus, the relative success of the active
pump in preventing rhodamine 123 uptake could be
due to its low affinity towards membranes and its slow
transmembrane passive transport [14]. On the other
hand, the relatively minor success of the efflux mecha-
nism in preventing TMR uptake into the cells could be
due to the high affinity of this dye to membranes and
its fast transmembrane passive transport [14]. A con-
clusion from our experiments is that, whether in the
presence or the absence of active transport into the
mitochondria, the free concentration of rhodamines in

the cytoplasm of the cells remains significantly lower
compared to their extracellular concentration. More-
over, the role played by mitochondria in allowing an
increased uptake of rhodamines into MDR ‘sensitive’
cells suggests that the efficiency of MDR in preventing
the cellular uptake of hydrophobic agents depends on
two competitions: (a) competition between the active
pumping of the agents out of the cells and their pas-
sive uptake [10,31] and (b) competition between the
active pumping of the agents out of the cells and their
intracellular binding to receptors and ⁄ or pumping into
intracellular compartments.
Because the rhodamines were excluded from all cell
lines tested, it is reasonable to assume that the NBD-
Cl-sensitive pump mediating this efflux is ubiquitous in
cell lines, and probably also in malignant tumors and
healthy tissues. The inhibition pattern of the NBD-Cl-
sensitive efflux suggests that it might be mediated by a
member of the ABC transporter family. The efflux is
inhibited by ATP depletion; by vanadate, which has
been shown to inhibit Pgp, MRP1 and MRP2 [32–34];
by MK571, which has been shown to inhibit MRP1,
MRP2, MRP4 and MRP7 [26,27,35,36]; and by indo-
methacin, which has been shown to inhibit MRP1,
MRP3, MRP4 and MRP6 [37–40]. Of these transport-
ers, MRP1 appears to be the best candidate to mediate
the innate efflux from ‘sensitive’ cells for several
reasons: (a) it is widely distributed in cell lines and
tissues [4]; (b) it is expressed in all the cell lines tested
here; (c) MRP1 is inhibited by NBD-Cl; and (d) the

efflux kinetics of rhodamines from NBD-Cl-inhibited
MRP1 over-expressing cells are similar to those exhib-
ited by ‘sensitive’ cells. Thus, the MRP1 levels present
in MDR ‘sensitive’ cells are sufficient to prevent equili-
bration throughout the cells of rhodamines, and
probably also of hydrophobic anticancer drugs, such
as doxorubicin.
As mentioned above, the data obtained in the pres-
ent study suggest that MRP1 interferes with the accu-
mulation of rhodamines and drugs participating in the
MDR phenomenon inside a wide variety of healthy
and malignant cells. Presumably, drugs exhibiting a
high affinity toward their intracellular targets over-
come this effect of MRP1 by tight binding to their
receptors. Thus, the resistance provided by the over-
expression of ABC transporters, such as Pgp, to MDR
cells could be due to their ability to compete with the
tight binding of the drugs to their respective targets,
rather than their capacity to compete with the passive
uptake of the drugs across the plasma membrane. It is
possible that the capacity of MRP1 to reduce the
intracellular concentrations of certain drugs in ‘sensi-
tive’ cells is more relevant to the clinical side-effects of
the drugs and less relevant to their chemotherapeutic
effect upon binding to their specific targets.
Experimental procedures
K562, a human leukemia cell line established from a
patient with chronic myelogeneous leukemia in blast trans-
formation [41], was purchased from ATCC (Rockville,
MD, USA) and maintained in RPMI medium (Biological

Industries, Beit-Haemmek, Israel). The K562 Pgp over-
expressing subline was obtained by sequential exposure of
cells to increasing concentrations of doxorubicin and was
maintained in the presence of 0.5 lm doxorubicin. A total
of 2008 parental cells and their MRP1, MRP2 and MRP3
over-expressing cell lines, as well as HEK293 parental cells
and HEK293 over-expressing MRP4 [42] or MRP5 [43],
were kindly provided by P. Borst (Netherlands Cancer
Institute, Amsterdam, the Netherlands) and grown in
RPMI-1640 (Sigma-Aldrich, Rehovot, Israel). The CIR
[44], CCRF-CEM [45] and the Jurkat [46] cell lines were
purchased from ATCC (Rockville, MD, USA) and main-
tained in RPMI-1640 (Sigma-Aldrich). GLC4 cells and
MRP1 over-expressing GLC4 ⁄ ADR cells [47] were
cultured in RPMI 1640. All media were supplemented with
10% fetal bovine serum, 100 IUÆmL
)1
penicillin and
100 lgÆmL
)1
streptomycin (Invitrogen, Rehovot, Israel)
and the cells were grown at 37 °C under 5% CO
2
⁄ humi-
dified air.
D. Yeheskely-Hayon et al. Innate multidrug resistance to rhodamines
FEBS Journal 276 (2009) 637–648 ª 2008 The Authors Journal compilation ª 2008 FEBS 645
TMRM, rhodamine 123, rhodamine 6G, silicone oil
AR200, NBD-Cl and mineral oil were purchased from
Sigma. TMR was purchased from Molecular Probes (Invi-

trogen).
For determination of the amount of rhodamine associ-
ated with cells, cells were incubated with the dyes in a
medium containing NaCl (132 mm), KCl (3.5 mm), CaCl
2
(1 mm), MgCl
2
(0.5 mm), glucose (10 mm), CCCP (1 lm)
and Hepes–Tris buffer (20 mm, pH 7.4). Samples contain-
ing 4 · 10
5
cells in 0.4 mL of medium were withdrawn
and placed in an Eppendorf-style microfuge above a
0.2 mL cushion composed of 95 parts silicone oil AR 200
(d
20
= 1.049) and five parts mineral oil (d
20
= 0.89). After
centrifugation for 4 min at 13 226 g at room temperature,
the oil cushion was washed three times with water by
suction. Subsequently, all of the upper phase, including
part of the oil cushion, was removed, leaving a fraction of
the oil above the cell pellets. The cell pellets were dissolved
by addition of 0.1 mL of guanidine HCl (5 m) buffered
with Hepes–Tris (50 mm, pH 7.4), centrifugation for
5 min, and incubation for at least 1 h at room tempera-
ture. The dissolved samples were mixed thoroughly with
0.5 mL of water and centrifuged for 5 min. Samples
(0.4 mL) were withdrawn from the pellets dissolved in the

aqueous phase. The fluorescence of TMR, TMRM, rhoda-
mine 123 and rhodamine 6G was determined using an
excitation wavelength of 552, 563, 496 or 527 nm and an
emission wavelength of 576, 583, 526 or 552 nm, respec-
tively. To ensure fidelity of the assay, dye-free cell samples
were mixed with known amounts of rhodamines and pro-
cessed as above. The rhodamine yield, thus obtained,
matched the amount expected. To determine the volume
of incubation medium carried through the oil cushion
together with the cells, a cell sample was incubated on ice
with 10 lm acidic dye (calcein) and processed as above.
The amount of calcein associated with the cells was equiv-
alent to < 0.05% of the sample volume. The time period
required to separate cells from the external medium was
equivalent to 1.5 min. All curves were adjusted accord-
ingly.
For fluorescence microscopy, K562 cells and 2008
attached cells were grown on poly-l-lysine coated 35 mm
plates (Ibidi GMBH, Mu
¨
nchen, Germany) and on uncoated
plates, respectively. The cells were washed twice with
NaCl ⁄ P
i
, prior to their incubation with the dye and inhibi-
tors. Cell images were photographed using an Axiovert 200
inverted fluorescent microscope (Carl Zeiss, Oberkochen,
Germany) equipped with orca-ER HDcam (Hamamatsu,
Japan) and a ·40 objective.
References

1 Juliano RL & Ling V (1976) A surface glycoprotein
modulating drug permeability in Chinese hamster ovary
cell mutants. Biochim Biophys Acta 455, 152–162.
2 Chen CJ, Chin JE, Ueda K, Clark DP, Pastan I, Got-
tesman MM & Roninson IB (1986) Internal duplication
and homology with bacterial transport proteins in the
mdr1 (P-glycoprotein) gene from multidrug-resistant
human cells. Cell 47, 381–389.
3 Gros P, Croop J & Housman D (1986) Mammalian
multidrug resistance gene: complete cDNA sequence
indicates strong homology to bacterial transport pro-
teins. Cell 47, 371–380.
4 Borst P & Elferink RO (2002) Mammalian ABC trans-
porters in health and disease. Annu Rev Biochem 71,
537–592.
5 Szakacs G, Paterson JK, Ludwig JA, Booth-Genthe C
& Gottesman MM (2006) Targeting multidrug resis-
tance in cancer. Nat Rev Drug Discov 5, 219–234.
6 Cole SP, Bhardwaj G, Gerlach JH, Mackie JE, Grant
CE, Almquist KC, Stewart AJ, Kurz EU, Duncan AM
& Deeley RG (1992) Overexpression of a transporter
gene in a multidrug-resistant human lung cancer cell
line. Science 258, 1650–1654.
7 Doyle LA, Yang W, Abruzzo LV, Krogmann T, Gao
Y, Rishi AK & Ross DD (1998) A multidrug resistance
transporter from human MCF-7 breast cancer cells
[Published erratum appears in Proc Natl Acad Sci USA
1999; 96: 2569]. Proc Natl Acad Sci USA 95, 15665–
15670.
8 Miyake K, Mickley L, Litman T, Zhan Z, Robey R,

Cristensen B, Brangi M, Greenberger L, Dean M, Fojo
T et al. (1999) Molecular cloning of cDNAs which are
highly overexpressed in mitoxantrone-resistant cells:
demonstration of homology to ABC transport genes.
Cancer Res 59 , 8–13.
9 Allikmets R, Schriml LM, Hutchinson A, Romano-
Spica V & Dean M (1998) A human placenta-specific
ATP-binding cassette gene (ABCP) on chromosome
4q22 that is involved in multidrug resistance. Cancer
Res 58, 5337–5339.
10 Eytan GD & Kuchel PW (1999) Mechanism of action
of P-glycoprotein in relation to passive membrane per-
meation. Int Rev Cytol 190, 175–250.
11 Stein WD (1997) Kinetics of the multidrug transporter
(P-glycoprotein) and its reversal. Physiol Rev 77, 545–
590.
12 Wielinga PR, Westerhoff HV & Lankelma J (2000) The
relative importance of passive and P-glycoprotein medi-
ated anthracycline efflux from multidrug-resistant cells.
Eur J Biochem 267, 649–657.
13 Mankhetkorn S, Dubru F, Hesschenbrouck J, Fiallo M
& Garnier-Suillerot A (1996) Relation among the
resistance factor, kinetics of uptake, and kinetics of the
P-glycoprotein-mediated efflux of doxorubicin, daun-
orubicin, 8-(S)-fluoroidarubicin, and idarubicin in multi-
drug-resistant K562 cells. Mol Pharmacol 49, 532–539.
14 Loetchutinat C, Saengkhae C, Marbeuf-Gueye C &
Garnier-Suillerot A (2003) New insights into the P-gly-
Innate multidrug resistance to rhodamines D. Yeheskely-Hayon et al.
646 FEBS Journal 276 (2009) 637–648 ª 2008 The Authors Journal compilation ª 2008 FEBS

coprotein-mediated effluxes of rhodamines. Eur J Bio-
chem 270, 476–485.
15 Schinkel AH, Smit JJ, van Tellingen O, Beijnen JH,
Wagenaar E, van Deemter L, Mol CA, van der Valk
MA, Robanus-Maandag EC, te Riele HP et al. (1994)
Disruption of the mouse mdr1a P-glycoprotein gene
leads to a deficiency in the blood–brain barrier and to
increased sensitivity to drugs. Cell 77, 491–502.
16 Lankas GR, Wise LD, Cartwright ME, Pippert T &
Umbenhauer DR (1998) Placental P-glycoprotein defi-
ciency enhances susceptibility to chemically induced
birth defects in mice. Reprod Toxicol 12, 457–463.
17 Sparreboom A, van Asperen J, Mayer U, Schinkel AH,
Smit JW, Meijer DK, Borst P, Nooijen WJ, Beijnen JH
& van Tellingen O (1997) Limited oral bioavailability
and active epithelial excretion of paclitaxel (Taxol)
caused by P-glycoprotein in the intestine. Proc Natl
Acad Sci USA 94, 2031–2035.
18 Kamimoto Y, Gatmaitan Z, Hsu J & Arias IM (1989)
The function of Gp170, the multidrug resistance gene
product, in rat liver canalicular membrane vesicles.
J Biol Chem 264, 11693–11698.
19 Johnson DR, Finch RA, Lin ZP, Zeiss CJ & Sartorelli
AC (2001) The pharmacological phenotype of combined
multidrug-resistance mdr1a ⁄ 1b- and mrp1-deficient
mice. Cancer Res 61, 1469–1476.
20 Schinkel AH, Mayer U, Wagenaar E, Mol CA, van
Deemter L, Smit JJ, van der Valk MA, Voordouw AC,
Spits H, van Tellingen O et al. (1997) Normal viability
and altered pharmacokinetics in mice lacking mdr1-type

(drug-transporting) P-glycoproteins. Proc Natl Acad Sci
USA 94, 4028–4033.
21 Lorico A, Rappa G, Finch RA, Yang D, Flavell RA &
Sartorelli AC (1997) Disruption of the murine MRP
(multidrug resistance protein) gene leads to increased
sensitivity to etoposide (VP-16) and increased levels of
glutathione. Cancer Res 57, 5238–5242.
22 Maliepaard M, Scheffer GL, Faneyte IF, van Gastelen
MA, Pijnenborg AC, Schinkel AH, van De Vijver MJ,
Scheper RJ & Schellens JH (2001) Subcellular localiza-
tion and distribution of the breast cancer resistance
protein transporter in normal human tissues. Cancer
Res 61, 3458–3464.
23 Chen LB (1989) Fluorescent labeling of mitochondria.
Methods Cell Biol 29, 103–123.
24 Marbeuf-Gueye C, Broxterman HJ, Dubru F, Priebe W
& Garnier-Suillerot A (1998) Kinetics of anthracycline
efflux from multidrug resistance protein-expressing
cancer cells compared with P-glycoprotein-expressing
cancer cells. Mol Pharmacol 53, 141–147.
25 al-Shawi MK & Senior AE (1993) Characterization of
the adenosine triphosphatase activity of Chinese ham-
ster P-glycoprotein. J Biol Chem 268, 4197–4206.
26 Chen ZS, Hopper-Borge E, Belinsky MG, Shchaveleva
I, Kotova E & Kruh GD (2003) Characterization of the
transport properties of human multidrug resistance pro-
tein 7 (MRP7, ABCC10). Mol Pharmacol 63, 351–358.
27 Gekeler V, Ise W, Sanders KH, Ulrich WR & Beck J
(1995) The leukotriene LTD4 receptor antagonist
MK571 specifically modulates MRP associated multi-

drug resistance. Biochem Biophys Res Commun 208,
345–352.
28 van Aubel RA, van Kuijck MA, Koenderink JB,
Deen PM, van Os CH & Russel FG (1998) Adeno-
sine triphosphate-dependent transport of anionic con-
jugates by the rabbit multidrug resistance-associated
protein Mrp2 expressed in insect cells. Mol Pharmacol
53, 1062–1067.
29 Feller N, Broxterman HJ, Wahrer DC & Pinedo HM
(1995) ATP-dependent efflux of calcein by the multi-
drug resistance protein (MRP): no inhibition by intra-
cellular glutathione depletion. FEBS Lett 368, 385–388.
30 Rabindran SK, He H, Singh M, Brown E, Collins KI,
Annable T & Greenberger LM (1998) Reversal of a
novel multidrug resistance mechanism in human colon
carcinoma cells by fumitremorgin C. Cancer Res 58,
5850–5858.
31 Eytan GD, Regev R, Oren G & Assaraf YG (1996) The
role of passive transbilayer drug movement in multidrug
resistance and its modulation. J Biol Chem 271, 12897–
12902.
32 Horio M, Gottesman MM & Pastan I (1988) ATP-
dependent transport of vinblastine in vesicles from
human multidrug-resistant cells. Proc Natl Acad Sci
USA 85, 3580–3584.
33 Qian YM, Qiu W, Gao M, Westlake CJ, Cole SPC
& Deeley RG (2001) Characterization of binding of
leukotriene C4 by human multidrug resistance protein
1. Evidence of differential interactions with NH2-and
COOH-proximal halves of the protein. J Biol Chem

276, 38636–38644.
34 Bakos E, Evers R, Sinko E, Varadi A, Borst P & Sark-
adi B (2000) Interactions of the human multidrug resis-
tance proteins MRP1 and MRP2 with organic anions.
Mol Pharmacol 57, 760.
35 Aubel R, Kuijck MA, Koenderink JB, Deen PMT, Os
CH & Russel FGM (1998) Adenosine triphosphate-
dependent transport of anionic conjugates by the rabbit
multidrug resistance-associated protein Mrp2 expressed
in insect cells. Mol Pharmacol 53, 1062.
36 Reid G, Wielinga P, Zelcer N, De Haas M, Van Deem-
ter L, Wijnholds J, Balzarini J & Borst P (2003) Char-
acterization of the transport of nucleoside analog drugs
by the human multidrug resistance proteins MRP4 and
MRP5. Mol Pharmacol 63, 1094–1103.
37 Evers R, de Haas M, Sparidans R, Beijnen J, Wielinga
PR, Lankelma J & Borst P (2000) Vinblastine and sul-
finpyrazone export by the multidrug resistance protein
MRP2 is associated with glutathione export. Br J
Cancer 83, 375–383.
D. Yeheskely-Hayon et al. Innate multidrug resistance to rhodamines
FEBS Journal 276 (2009) 637–648 ª 2008 The Authors Journal compilation ª 2008 FEBS 647
38 Bodo A, Bakos E, Szeri F, Varadi A & Sarkadi B
(2003) Differential modulation of the human liver
conjugate transporters MRP2 and MRP3 by bile
acids and organic anions. J Biol Chem 278, 23529–
23537.
39 Reid G, Wielinga P, Zelcer N, van der Heijden I, Kuil
A, de Haas M, Wijnholds J & Borst P (2003) The
human multidrug resistance protein MRP4 functions as

a prostaglandin efflux transporter and is inhibited by
nonsteroidal antiinflammatory drugs. Proc Natl Acad
Sci USA 100, 9244–9249.
40 Ilias A, Urban Z, Seidl TL, Le Saux O, Sinko E, Boyd
CD, Sarkadi B & Varadi A (2002) Loss of ATP-depen-
dent transport activity in pseudoxanthoma elasticum-
associated mutants of human ABCC6 (MRP6). J Biol
Chem 277, 16860–16867.
41 Lozzio CB & Lozzio BB (1975) Human chronic myelog-
enous leukemia cell-line with positive Philadelphia chro-
mosome. Blood 45, 321–334.
42 Adachi M, Sampath J, Lan LB, Sun D, Hargrove P,
Flatley R, Tatum A, Edwards MZ, Wezeman M,
Matherly L et al. (2002) Expression of MRP4 confers
resistance to ganciclovir and compromises bystander
cell killing. J Biol Chem 277, 38998–39004.
43 Wijnholds J, Mol CA, van Deemter L, de Haas M,
Scheffer GL, Baas F, Beijnen JH, Scheper RJ, Hatse S,
De Clercq E et al. (2000) Multidrug-resistance protein 5
is a multispecific organic anion transporter able to
transport nucleotide analogs. Proc Natl Acad Sci USA
97, 7476–7481.
44 Storkus WJ, Alexander J, Payne JA, Dawson JR &
Cresswell P (1989) Reversal of natural killing suscepti-
bility in target cells expressing transfected class I HLA
genes. Proc Natl Acad Sci USA 86, 2361–2364.
45 McCarthy RE, Junius V, Farber S, Lazarus H & Foley
GE (1965) Cytogenetic analysis of human lymphoblasts
in continuous culture. Exp Cell Res 40, 197–200.
46 Weiss A, Wiskocil RL & Stobo JD (1984) The role of

T3 surface molecules in the activation of human T cells:
a two-stimulus requirement for IL 2 production reflects
events occurring at a pre-translational level. J Immunol
133, 123–128.
47 Zijlstra JG, de Vries EG & Mulder NH (1987) Multi-
factorial drug resistance in an adriamycin-resistant
human small cell lung carcinoma cell line. Cancer Res
47, 1780–1784.
48 Davey RA, Longhurst TJ, Davey MW, Belov L, Harvie
RM, Hancox D & Wheeler H (1995) Drug resistance
mechanisms and MRP expression in response to epiru-
bicin treatment in a human leukaemia cell line. Leuk
Res 19, 275–282.
49 Beckmann R, Smythe JS, Anstee DJ & Tanner MJ
(1998) Functional cell surface expression of band 3, the
human red blood cell anion exchange protein (AE1), in
K562 erythroleukemia cells: band 3 enhances the cell
surface reactivity of Rh antigens. Blood 92 , 4428–4438.
Innate multidrug resistance to rhodamines D. Yeheskely-Hayon et al.
648 FEBS Journal 276 (2009) 637–648 ª 2008 The Authors Journal compilation ª 2008 FEBS