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

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

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-

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

)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

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

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

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

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

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.

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

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

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

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

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

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