d~malaf
~OI.OOY
B:BIOLOGY
ELSEVIER
Journal of Photochemistry and Photobiology B: Biology 34 (1996) 177-182
Intracellular pH does not affect drug extrusion by P-glycoprotein
Katalin Goda a, L~iszl6 Balkay b, Ter6z M&i~in b, Lajos Tr6n b, Adorj~in Aszal6s c,
G~ibor Szab6 Jr. a,,
a Department of Biophysics, b PET Center, University Medical School of Debrecen. 4012 Debrecen, Hungary
c Division of Research and Testing. CDER HFD-471, Food and Drug Administration, 200 C St. SW, Washington, DC 20204, USA
Received 17 August ! 995; accepted 13 November 1995
Abstract
The intracellular pH (pHi) of cells exhibiting multidrug resistance (MDR) related to the expression of the P-glycoprotein (Pgp) is often
more alkaline than that of the parental cells, as also observed for the KB-V I/KB-3-1 system in this paper. The possible role of an elevated
pHi in Pgp-related MDR has been investigated by shifting back the pHi of the MDR ÷ cells to a more acidic value using the mobile proton
ionophore carbonylcyanide m-chlorophenylhydrazone (CCCP). The influence of CCCP-evoked ApHi on relative daunorubicin (DNR)
accumulation was similar in the ease of several Pgp positive and negative cell lines, in view of flow cytometric and radioactive drug
accumulation studies and measuring DNR levels in the medium in a flow-through system. Our data argue against a significant effect of pHi
on Pgp pumping efficiency. However, an indirect connection between pHi regulation and the MDR phenotype is suggested by the fact that
acidification of the external medium in the presence of verpamil could be observed exclusively in MDR + cells.
Keywords: Multidrug resistance; Intraceilular pH; P-glycoprotein
1. Introduction
Intracellular levels of supravital fluorescent drugs are controlled by various cellular parameters, including active drug
export mechanism, e.g. the membrane pumps related to the
multidrug resistance (MDR) phenomenon. One of these proteins, P-glycoprotein (Pgp; PI70), is considered as one of
the main mechanisms acquired by cancer cells that prevent
effective chemotherapy (for reviews see Refs. [ 1,2] ). This
170 kDa transport ATPase extrudes its substrates against a
concentration gradient [ 3]. Based on sequence homologies
and functional analogies, this pump glycoprotein belongs to
a group of transport proteins that transfer hydrophobic molecules, peptides and various drugs across the cell membrane
[4--6]. The drug-resistant phenotype can be functionally
reversed by a wide range of chemicals, including calcium
channel blockers [7], calmodulin inhibitors [2]2 lysosomotropic agents [8], some ionophores [9], etc., all of which
greatly increase drug accumulation in P170-expressing cells.
Competition for the substrate binding site (s) [ 2,7 ] and aUosteric coupling [ 10] have been suggested to account for the
mechanism of this reversion.
*Corresponding author.
I 0 ! 1- 1344/96/$15.00 © ! 996 Elsevier Science S.A. All rights reserved
SSD! 1011 - 1344 ( 95 ) 0 7 2 8 2 - 9
P170 substrates (as well as reversing agents) are usually
amphiphilic compounds [ 11]. In view of their pH-dependent
lipophilicity [ 12], the intracellular pH (pHi) may have a
decisive role in the exit process mediated by PI70. pHi may
influence drug partitioning between the cell and its environment and/or between the various subcellular compartments
[ 13,14]. A large body of data suggest that the pHi of multidrug-resistant cell lines appears to be higher than that of their
non-resistant counterparts [15-20]. The elevated pHi of
resistant cells has been interpreted in terms of ( 1 ) a transport
mechanism specific to protonated substrates [18], (2) a
direct contribution of an alkaline pHi to the efficiency of the
pump-m~dia:ed drug extrusion [ 21 ], (3) Pgp-generated
changes in pHi and qt [20] that could be responsible for
decreased drug accumulation and (4) Pgp-unrelated mechanisms [ 19,21 ].
To examine the above alternatives, we measured the drug
accumulation of various resistant and sensitive cell Jines as a
function of pHi, using daunorubicin (DNR), a fluorescent,
DNA-binding, anticancer drug [22], and P170 substrate.
Simon et al. [ 19] and Altenberg et al. [21 ] also examined
the effect of pHi alterations on drug accumulation, supporting
the last interpretation above. In view of the complexity of
parameters possibly influencing the outcome of such studies
178
K. Goda et al. / Journal of Photochemistry and Photobiology B: Biology 34 (1996~ 177-182
(e.g. dependence of fluorescence quantum yield on drug compartmentalization [23], alteration of the HCO3-/CIexchange implicated in relation to the MDR phenotype [ 24 ]
by the treatments used), we applied various methods complementing each other to address the above possibilities. In
our experiments, pHi was acidified by the mobile proton
ionophore
carbonylcyanide m-chlorophenyihydrazone
(CCCP) [25] applied at low extracellular potassium concentration ([Ko + ]), when an inside negative membrane
potential drives the protons, carried by CCCP, into the cell.
The flow cytometric drug accumulation measurements are
sensitive to population heterogeneities and 3H-DNR uptake
data are not biased by the possible changes in DNR fluorescence quantum yield. The flow-through system was also
applied for getting information about the immediate effects
of ionophore treatment; since it is not influenced by possible
spectral effects, it is expected to be more sensitive than the
flow cytometric detection system. In addition, the changes in
extracellular pH (pHo) were also monitored in response to
incubation of the resistant and sensitive cells with various
Pgp substrates.
2. Materials and methods
2.1. Chemicals and reagents
Cells were trypsinized 2 days prior to the experiments and
maintained without vinblastine. In some experiments the
A2780/2780 Av (human ovarian carcinoma [27] ) cell pair
was also used. The cells were checked for mycoplasma by
the mycroplasma T.C. rapid detection system with a 3Hlabelled DNA probe from Gen-Probe Inc. and were found to
be negative.
2.3. Drug uptake measurements by flow cytometry
The "140 mM K +'' buffer contained 140 mM KCI, 10
mM NaCI, 10 mM Na-Hepes and 5 mM glucose (pH 7.4 at
room temperature). In the "2 mM K +'' buffer most of the
KC! was isotonically replaced by NaCI (or by choline chloride, with identical results). Cells were resuspended in the
appropriate buffer (at 4 × 105 cells mi-~) containing DNR
(3.55/~M) and incubated for 40 min at 37 °C. The ionophore
CCCP was added to the cells at a concentration of 25 or 50
/zM, simultaneously with DNR. The viability of cells after
incubations was checked by propidium iodide staining [ 28 ].
Drug uptake and pHi were determined using a Becton Dickinson FACS Star Plus flow cytometer equipped with a Spectra
Physics 164-08 argon ion laser. The fluorescence signal was
gated on the forward angle light scatter (FSC) signal to
exclude the dead cells and cell debris from the analysis. The
argon ion laser was tuned to 488 nm and used at a powe~~of
500 mW. Emission was detected through a 540 nm broad
band interference filter (IF) and a 620 nm Iongpass filter (for
DNR).
BCECF-AM (tetraacetoxymethylester of the dye 2',7bis(2-carboxyethyl)-5(and 6).-carboxyfluorescein) was
obtained from Molecular Probes Inc. (Eugene, USA). Daunorubicin (DNR), rhodamine 123 (R123), CCCP (carbonylcyanide m-chlorophenylhydrazone), nigericin, valinomycin (Vai), verapamil (Ver), vinblastine (Vbl) and inorganic chemicals were provided by Sigma-Aldrich (Budapest, Hungary).
Hepes
(N-[2-hydroxyethyl]piperazine-N'-[ethanesulphonic acid]), PSC833 and [G-3H]daunorubicin/HC! (specific activity 1.6 Ci mmol-~) were
purchased from SERVA (Heidelberg, Germany), Sandoz
( Basle, Switzerland) and Dupont de Nemours ( 's-Hertogenbosch, Netherlands) respectively. Cyclosporin A was FDA
standard (A. Aszal6s).
The above buffer solutions were used supplemented with
5% foetal calf serum. The samples contained 0.3 × l0 s cells
ml- ~.The cells were incubated in the presence of I pM DNR
(containing 2 nM 3H-DNR) and CCCP or reversing agents
(at concentrations indicated in Fig. 3) at 37 °C. After incubation the cells were washed twice with ice-cold buffer solution. The cell pellets were transferred to Opti-phase Ill (LKB,
Bromma, Sweden) scintillation liquid and then the accumulated 3H-DNR was measured by scintillation counting.
2.Z Cell lines
2.5. Flow-through experiments
The drug-sensitive human epidermoid carcinoma cell line
KB-3-1 and its vinblastine-selected multidrug-resistant variant KB-V 1 ( isolated from KB-3-1 by stepwise selection with
increasing vinblastine concentrations [26]) were used in
most of the measurements. These cell lines were grown as
monolayer cultures at 37 °C in an incubator containing ';%
CO2 and 95% air and maintained by regular passage in Dulbecco's minimal essential medium (supplemented with 10%
heat-inactivated foetal calf serum, 2 mM L-glutamine, 100
units m l - ~penicillin and 100/zg ml- ! streptomycin). KBV 1 cells were cultured in the presence of 180 nM vinblastine.
The flow-through system is described in detail by Lankelma et ai. [29]. Briefly, a monolayer of approximately
1 × 107 cells attached to a glass chamber (surface area 50
cm2, height 0.1 mm) was perfused with the appropriate buffer
containing I or 2/.tM DNR, at a constant flow rate of 200/tl
rain- ~at 37 °C, until a steady state level was reached. Pulse
injections (15 ~!) of reversing agents (verpamil) or ionophores (CCCP) were introduced into the flowing perfusion
medium using a high performance liquid chromatography
(HPLC) injection valve. Changes in the 480-560 nm fluorescence were measured on-line in the perfusion medium at
2.4. ~H-DNR accumulation experiments
179
K. Goda et al. / Journal of Photochemistry and Photobiology B: Biology 34 (1996) 177-182
Table 1
Changes in pHi (A pHi) after ionophore treatment of KB-3- ! ( M D R - ) and
KB-V! (MDR + ) cells, pHi was measured by flow cytometry (see Section
2) at 2 or 140 mM [Ko + ] and pHo= 7.4. Means + SEM were calculated
from three independent experiments. CCCP and Vai were used at 50 and 9
/tM concentration respectively
[Ko + ] (mM)
Cell line
CCCP
CCCP+Val
2
KB-3-1
KB-V- !
- 0.40 ___0.08
- 0.42 4- 0.06
- 0.68 + 0.06
- 0.81 + 0.02
140
KB-3- l
KB-V- 1
- 0.09 + 0.06
0.03 4- 0.03
0.02 4- 0.02
0.05 + 0.01
the outlet of the flow-through system, by a fluorescence detector (type 821-FP, Jasco, Haschioji City, Japan).
2.6. pH~ and pH,, measurements
For measurement of pHi, cells (1 × 107 m l - ' ) in phosphate-buffered saline (PBS; 150 mM NaCl, 3.3 mM KCI, 8.6
mM Na2HPO4 × 12H20, 1.69 mM KH2PO4, pH 7.4) were
loaded with 10/.~M (MDR- cells) or 15 pM (MDR ÷ cells)
BCECF for 1 h at 37 °C. Calibration of pHi vs. fluorescence
was carried out using nigericin to set pHi to known values
[30]. pHi was determined within 5-10 min following ionophore treatment, by measuring the ratio of red and green
fluorescence intensities [ 31 ] (see Section 2.3).
For the measurement of changes in pHo upon pumping, the
cells (used at 1 x 106 m l - ' ) were incubated with various
MDR substrates in a "low K +'' buffer (see below), but
containing 0.8 mM Hepes, at 37 °C for 1 h. The pH of this
solution was stable without addition of cells. After incubation
the pH of supernatants was measured by a conventional pH
electrode.
was stable in time as measured after 30 min incubation. Acidification by CCCP occurred in a pHo-dependent manner, as
we could detect a larger pHi decrease at pHo = 6.9 (data not
shown). The addition of CCCP is expected to reduce pHi, the
protons being driven in by a negative inside membrane potenti~', (q/). This interpretation is also supported by the fact that
valinomycin (Val), which is expected to hyperpolarize the
membrane (as follows from the Goldman-Hodgkin-Katz
equation [33]), promoted acidification caused by CCCP.
Accordingly, in the "140 mM K +" buffer ( ~ = 0) the ionophores had only a minor effect on pHi (see Table 1).
The significant pHi differences demonstrated between
MDR ÷ and MDR- cells are in agreement with Refs. [ 1520] and in contrast with Refs. [ 34,35 ]. An increased pHi is
expected to decrease DNR accumulation independently from
Pgp, since the pH gradient is a strong driving force for (cellular) DNR uptake (towards the more acidic compartment;
see trapping of weak bases into liposomes by proton gradients
following the Henderson-Hasselbach rule [ 12,13 ] ). Were
protons to have a more specific role related to the Pgp pumping mechanism, the pHi would differentially affect MDR ÷
and MDR- cells.
3.2. Effect of CCCP treatment on DNR accumulation
3.2.1. Flow cytometric and 3H-DNR uptake studies
As Fig. 1 shows, KB-VI cells, without ionophore treatment, accumulated approximately five times less DNR than
the KB-3-1 cells. The DNR fluorescence of either KB-V1
(MDR) ÷ or KB-3-1 (MDR-) cells was usually decreased
to a minor extent (to 96.5%+19.7%; p < 0 . 1 ) by CCCP
(added simultaneously with DNR). Addition of CCCP to
MDR ÷ cells preloaded with DNR did not affect mean fluo350
i
3. Results and discussion
300
3.1. lntraceUular acidification by CCCP
250
For flow cytometric pHi measurements we used the
BCECF double-ratio method of Balkay et al. [31 ], which
excludes the possible artefacts related to the different BCECF
accumulation of MDR ÷ and MDR- cells [32] since the
measured fluorescence emission ratios (proportional to pHi;
see Section 2.6) are independent of the actual intracellular
dye concentration and cell volume. However, we rely only
on the changes in and relative values of pHi in our conclusions.
The pHi of untreated KB-3-1 (sensitive) cells was lower
than that of the KB-VI (resistant) line (by 0.23 + 0.02 (mean
+standard error of measurement ((SEM) pH unit; the difference was significant atp < 0.01, calculated from five independent experiments). As Table 1 shows, the addition of
CCCP acidified both the resistant and parental cells by 0.30.5 pH unit in the "2 mM K ÷'' buffer. This pHi decrease
.~
I
i
I
i * IB I
,
i
I
I
i I I I i
i
I
i
I
i T II
I
1
i
' [
I|
I|
II
:: ll
--
150
. "..
_
'"i"
_
50
ll
.. ,...
-
I
'
;11
f.*.•
-
..i
0
I0 o
10 i
10 2
10 3
Fluorescence intensity
Fig. 1. F!uorescence intensity distribution histograms of KB-V i and KB-31 cells incubated with 3.55 p M DNR for 30 rain. KB-V 1 cells were treated
with 50 pM Ver (full curve), 50/tM CCCP (broken curve) or were analysed
without these additions ( dotted curve ); lon~ dashes represent untreated KB3-1 cells.
180
K. Goda et al. /Journal of Photochemistry and Photobiology B: Biology 34 (1996) 177-182
140 m M K +
2 mM K*
A
1400
g~
1200
g~
1000
O
om
,tJ
_=
H
O
t
.=
4'
800 !
600
400
Z
A
r-
200
m j
0
2SIJMC
I
.
.
.
.
.
S0pMC 10pMPSC S0pMVer
2 rnM K +
140 mM K +
1400
i.......
1200
m
.2
1000
M
=
U
800
m
400
n
.
.
.
.
.
.
.
.
.
.
.
.
.
.
i
600
W
200
m
o
2$ltMC
S0~MC 10pMPSC S0pMVer
Fig. 2.3H-DNR accumulation of 2780 Al' ( MDR +, A) and A2780 ( MDR -,
B ) cell lines after 40 min incubation with !/xM DNR (containing 2 nM ~HDNR) in the presence of CCCP (C) or reversing agents (Ver, verapamil;
PSC, PSC833). Means of triplicate samples with SEM are shown (in the
case of one representative experiment out of three).
rescence levels (data not shown). No population heterogeneity emerged in either of these experiments as a consequence
of ionophore treatment (in addition to the revertant cells
present in some cultures). Similar results were obtained in
the case of A2780 (MDR-) and 2780 AD (MDR +) cells
(data not shown).
The flow cytometric data agree with the results of 3H-DNR
uptake measurements, as shown in Fig. 2. CCCP had no effect
on drug accumulation (measured after 40 min incubation
with the drug) in either MDR + or MDR- cells. The CCCPprompted acidification was stable in time as measured up to
30 min (data not shown). In spite of the relatively high
ionophore concentrations applied, the cells were alive in
terms of both membrane permeability (routinely checked in
flow cytometric experiments by propidium iodide staining)
and intracellularATP concentrations (greater than 2 mM, as
measured by ion exchange HPLC; data not shown). The
increase in DNR uptake evoked by verapamil (Ver) or the
cyclosporin A analogue PSC833 demonstrates the sensitivity
of our experimental systems (see Figs. 1 and 2A).
CCCP treatment enhances the proton permeability of intracellular membrane systems (besides decreasing pHi at low
[ Ko ÷ ] ), equilibrating the proton gradients in mitochondria
and lysosomes. The increment of lysosomal pH was immediate (and stable for at least 30 min) after the addition of 50
/.LM CCCP (at both low and high [ Ko ÷ ] ) and it was even
higher than that caused by 50 mM methylamine (our unpublished data obtained by the FITC-dextran method [36] ).
However, a significant contribution of the lysosomal compartment to cellular drug accumulation can be ruled out, since
alkalinization of the lysosomes by CCCP at 140 mM [ Ko ÷ ]
(when the pHi was unchanged; see Table 1) did not affect
3H-DNR accumulation (see Fig. 2).
3.2.2. Flow-through measurements
In contrast with the above data, a significant DNR influx
was detected into KB-V 1 and KB-3-1 cells immediately after
CCCP injection in the flow-through system, as shown in Fig.
3 (traces A and B), perhaps owing to a higher sensitivity of
this method. The superior sensitivity of this system over flow
cytometric detection is explained by the fact that most of the
DNR fluorescence is quenched when it binds to DNA [ 23 ].
Furthermore, the effect of pHi decrease seems to dominate
over the effect of increased lysosomal pHi on this time scale,
as demonstrated by Demant et al. [ 37 ]. Their kinetic analysis
of the possible roles of transmembrane pH gradients in intracellular antracycline accumulation showed that the change in
pHi from 7.4 to 6.9 was followed by a moderate (35%)
increase in steady state accumulation of DNR. When the pHi
decrease is accompanied by the equilibration of lysosomal
pH (as in our 3H-DNR accumulation and flow cytometric
experiments), the overall CCCP effect on drug accumulation
is expected to be even smaller. MDR ÷ and MDR- cells
exhibited similar responses to CCCP. No shift in medium
A
CCCP
. . . .
g
B
.
O
e~
.
.
C
ccc~
.
.
.
.
.
.
.
V.r
Sn~n
O.| pllg
time
Fig. 3. Effect of 50/~M CCCP on steady state accumulation of DNR in KB3-1 (MDR-) and KB-VI (MDR + ) cells, measured in the flow-through
system at 2 mM [Ko + ]. The agents listed below were injected into the
perfusion medium after steady state between intracelhlar and medium DNR
was attained. A decrease in the medium fluorescence of DNR is a consequence of the net celhlar DNR uptake, while an increase shows net efflux
of DNR. Trace A shows the effect of 50/xM CCCP on the steady state
accumulation of 2/~M DNR by KB-3-! cells. Traces B and C show the
effects of 5 0 / , M CCCP and 25/~M Ver (repeated five times, within 2.5
rain) on the accumulation of I/~M DNR by KB-Vi cells.
K. Goda et al. / Journal of Photochemistry and Photobiology B: Biology 34 (1996) 177-182
Table 2
ApHo after I h incubationofKB-3-1 (MDR-) and KB-VI (MDR+ ) cells
with various MDR substrates (or reversing agents) The means of three
independent experiments + SEM are shown in the case of Ver or CsA
treatment; the other results were reproduced once; R123, Vbl and CsA
designate rhodamin i 23, vinblastineand cyclosporinA respectively
MDR
substrate
Concentration
A pHo
A pHo
KB-V1
KB-3-1
RI23
13/~M
26/~M
0.02
0.04
0.09
0. ! 1
DNR
17.7 p,M
35.4 p,M
0.00
0.03
0.04
0.04
Vbl
5.45/~M
i 0.9/~M
0.03
- 0.07
- 0.05
- 0.06
16.4 p,M
- 0.07
- 0.08
Ver
CsA
50/~M
5 ttg ml- t
0.28 + 0.03
0.02 + 0.00
- 0.01 -I-0.02
0.04 + 0.02
181
dam, Netherlands), Ursula A. Germann and Michael M. Gottesman (Bethesda, USA) for important suggestions and
critical comments. The authors also acknowledge Paul
Noordhuis for measuring the intracellular ATP levels. The
muitidrug-resistant cell lines were kindly donated by Dr.
M.M. Gottesman and Jan Lankelma. This publication has
been sponsored by the US-Hungarian Science and Technology Joint Fund in cooperation between the Department of
Biophysics, University Medical School of Debrecen and the
Division of Research and Testing, Food and Drug Administration under Project JFNO 127. This work was also supported by OTKA fund T017592 and ETT grant T-01 449/
93. Certain parts of the work were done with the financial
support of the Hungarian-Dutch Association.
References
DNR was detected upon CCCP addition at 140 mM [K,, ÷ ]
(data not shown) where pHi was unchanged (see Table 1 ).
Thus a decreased pHi appears to facilitate drug accumulation
as expected [ 12. ! 3 ], but in a Pgp-independent manner.
If the Pgp-driven drug export were coupled to proton transport [ 18 ] or the activity of Pgp were dependent on pHi, it
would have been reflected in a major difference between the
drug accumulation of MDR ÷ and M D R - cells in response
to the acidification of pHi. However, this was not seen, in
agreement with the data of Altenberg et ai. [ 21 ], who could
not detect an alteration in the unidirectional efflux of R123
in response to pHi changes.
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pHo upon pumping [ 18]. However, the change in pH,, after
1 h incubation with DNR (35 /xM), R123 ( 2 6 / x M ) , vinblastine ( 16.4/.tM) or CsA ( 5 / z g ml - ~) did not exceed that
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MDR ÷ cell lines are often distinguished from their parental
cells by a higher pHi, as well as differential acidification in
response to Ver (see above), imply that the MDR ÷ phenotype frequently involves an altered pHi regulation.
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The authors thank Drs. S~indor Damjanovich (Debrecen,
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