ICANCERRESEARCH52, 17-23, January 1, 19921
Energy-dependent Processes Involved in Reduced Drug Accumulation in Multidrug
resistant Human Lung Cancer Cell Lines without P-Glycoprotein Expression'
Carolina H. M. Versantvoort,2 Henricus J. Broxterman, Herbert M. Pinedo, Elisabeth G. E. de Vries, Nicole Feller,
Catharina M. Kuiper, and Jan Lankelma
Department ofMedical Oncology, Free University Hospital, de Boelelaan 1117, 1081 HV Amsterdam (C. H. M. V., H. I. B., H. M. P., N. F., C. M. K., I. L.J, and
Department ofMedical Oncology,State University ofGroningen, Groningen[E. G. E. d. V.], The Netherlands
cell line, SW-i 573, by selection for resistance to doxorubicin
ABSTRACT
(3, 4). During early steps of the selection, an MDR cell line
Mechanisms contributing to reduced cytotoxic drug accumulation were
studied in two multidrug-resistant (MDR) human lung cancer cell lines
(SW-1573/2R120) without any detectable expression of the
mdrl gene was isolated, whereas sublines isolated at higher
concentrations of doxorubicin overexpressed Pgp. The cross
without P-glycoprotein expression. In these (non-small cell) SW-1573/
2R120 and (small cell) GLC4/ADR MDR cells, the steady-state accu
mulation of (‘4CJdaunorubicin
was 30 and 12%, respectively, of that in
the parent cells. When cells, at steady state, were permeabilized with
digitonin, the amount of daunorubicin binding increased only in the
resistance
This MDR phenotype, i.e., a broad cross-resistance spectrum
and reduced cellular drug levels, has been reported recently for
other cell lines that do not overexpress Pgp (5—10).The broad
spectrum of drugs to which these cells are resistant makes it
unlikely that alterations in a common target site are the primary
cause of the resistance. An enhanced, energy-dependent efflux
of daunorubicin has been reported for non-Pgp MDR HL-60/
Adr (human leukemia), HT1O8O/Dr4 (human fibrosarcoma),
and COR-L23/R (human large cell lung carcinoma) cells (6, 7,
10). Since the level of Pgp expression in lung cancer generally
is low (1 1), other resistance mechanisms may be of importance
mm) uptake rate of this drug. No difference in initial effiux rate of
daunorubicin from preloaded cells could be detected between sensitive
and resistant SW-1573 cells. However,daunorubicinwas extruded 5-fold
faster from GLC4/ADRcells than from the parental cells. In the presence
of the energy metabolism inhibitors sodiumazide and deoxyglucose,the
reduced daunorubicinaccumulationsin the SW-1573/2R120and GLC4/
ADR MDR cells were (almost) completelyreversed.The effects of these
inhibitors on drug uptake were already apparent during the earliest
measured time points (<15 s). Also, the enhancedeffiux of daunorubicin
from GLC4/ADR cells was inhibited. In ATP-depleted cells, the intra
cellular pH was loweredby —03units in resistant as well as in sensitive
cells. The lower intracellular pH, however, could not account for the
increase in daunorubicin accumulation in the resistant cells. Also, for
vincristine and etoposide, the increases in drug accumulation under
were more pronounced
in the resistant
cells resembled
(3).
1573/2R120and GLC4/ADR cells was accompaniedby a lowerinitial (2
conditions
of the SW-1573/2R120
be defective in accumulation of vincristine and daunorubicin
resistant cells. The reduced accumulation of daunorubicin in the SW
energy-deprived
spectrum
that of the Pgp-expressing sublines and the cells also proved to
in lung cancer. One such mechanism may be related to topo
isomerase II activity (12). In a human small cell lung carcinoma
cell line (GLC4/ADR), part ofthe resistance could be explained
by reduced topoisomerase II activities (13). The possibility of
alternative (non-Pgp) drug transport mechanisms operating in
SW
1573/2R120cells than in the parent SW-1573cells.These results suggest
that accumulationof drugs in the non-P-glycoproteinMDR human lung
carcinoma cell lines SW-1573/2R120 and GLC4/ADR is reduced by an
energy-dependentdrug export mechanismwhich prevents efficienttrans
MDR cells has not been adequately investigated until now.
We studied the presence of putative energy-dependent
drug
port of drug to the target. Since P-glycoprotein expression in lung tumors
transporters
is generally
non-Pgp-mediated MDR lung cancer cell lines. By permeabili
zation of the plasma membrane after steady-state accumulation
of daunorubicin, we could show that the SW-1573/2R120, but
not the parent cells, keep daunorubicin binding to the nuclear
DNA below equilibrium at the applied extracellular concentra
low, these MDR lung cancer cell lines can be used as a model
to study alternative mechanisms leading to multidrug resistance in this
tumor type.
INTRODUCTION
tion. Furthermore,
By exposure to cytotoxic drugs, tumor cells can acquire
resistance
to structurally
and
functionally
unrelated
for a plasma
membrane
glycoprotein
com
of Mr 170,000—
180,000 (Pgp). Pgp is thought to function as an energy-depend
ent drug efflux pump (1, 2). However, a series of MDR cell
lines has been derived from a human non-small
cell lung cancer
Received 7/3/91; accepted 10/17/91.
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 Supported
by the
Netherlands
Cancer
Foundation
(grant
IKA
89—11).
H.
MATERIALS
whom
requests
for
reprints
should
be addressed,
at Department
(Paris, France),
of Medical
abbreviations
used
are:
MDR,
multidrug
resistance
(resistant);
on
AND METhODS
vincristine
sulfate from Sigma Chemical
Co. (St. Louis,
MO), and VP-16 from Bristol Myers Co. (Weesp, The Netherlands).
Oncology, Free University Hospital, BR 232, Dc Boelelaan 1117, 1081 HV
Amsterdam, The Netherlands.
3 The
energy depletion
Chemicals. Daunorubicin hydrochloride was obtained from Specia
J.
B. is a fellowof the Royal Netherlands Academyof Arts and Sciences.
2 To
the effects of cellular
(5, 13)
daunorubicin transport in these cells were studied. Since dau
norubicin is a weak base, changes of intracellular pH, which
might be attributed to alterations in membrane transport, were
taken into account. Energy-dependent transport of vincristine
and VP-16 was studied in the SW-1573 cells. From the exper
iments reported here, it appears that an energy-dependent
mechanism leading to decreased drug accumulation is operative
in non-Pgp-mediated MDR SW-i 573/2R1 20 and GLC/ADR
cells.
pounds. In vitro the cellular pharmacological basis for MDR3
often appears to be a reduced steady-state accumulation of
drugs caused by the overexpression of the mdrl gene, which
encodes
in SW-i 573/2R1 20 (3) and GLC4/ADR
BCECF-acetoxymethylesterwas from Molecular Probes (Eugene, OR).
HEPES,
Sodium
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid; PBS, phosphate-buffered
saline (136 mM NaCI, 2.5 m@iKCI, 6.6 m@iNa2HPO4, 1.5 m@iKH2PO4); Pgp,
P-glycoprotein; BCECF, 2'7'-bis-(2-carboxyethyl)-5 (and -6)carboxyfluorescein;
azide
was obtained
from
Baker
Chemicals
(Deventer,
The
Netherlands), and nigericin and 2-deoxy-d-glucose was from Sigma.
114-'4ClDaunorubicin
VP-16, etoposide.
(specific activity,
45 Ci/mol)
and IG-3Hlvincris
tine sulfate (specific activity, 4.8 Ci/mmol) were obtained from Amer
17
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1992 American Association for Cancer Research.
REDUCED DRUG ACCUMULATION NOT MEDIATED BY Pgp
sham (Amersham, United Kingdom); [G-3HIVP-16(specificactivity, I
Ci/mmol) was from Moravek Biochemicals(Brea, CA).
Cells. The human non-small cell lung carcinoma cell line SW-l573,
the pH sensitive, fluorescent dye BCECF (18). Cell suspensions in
medium A (1 x l0@cells/mi) were loaded for 30 mm at 37T with 5
originally
zM BCECF-acetoxymethylester.
isolated by Dr. A. Leibovitz
Intracellular pH Measurements. Intracellular pH was measured with
(Scott and White Clinic, Temple,
Cells were washed
and fluorescence
TX), and the doxorubicin-resistant subline SW-l573/2R120 have been
was recorded at 532 nm (emission) and 438 and 506 nm (excitation
described elsewhere (3, 14). The cells were cultured at 37C in 5—6% wavelengths). The ratio of the fluorescence measured with excitation
CO2in a humifiedincubatorin Dulbecco'smodifiedEagle'sessential wavelengthsset at 506 nm to the fluorescence measured at 438 nm was
medium (Flow Laboratories, Irvine, Scotland) supplemented with 7.5%
used to calculate the intracellular pH (19). The calculation was based
heat-inactivated fetal calf serum (GIBCO Europe, Paisley, Scotland)
on the ratios obtained by resuspending the cells in high K@buffer (145
and 20 mM HEPES (Serva, Heidelberg, Germany). The human small
mM KC1, 5 mM NaC1, 1 m@iCaC12, 1 mM MgCl2, 10 mM HEPES)
cell lung carcinoma cell line GLC4 and its doxorubicin-resistant subline
GLC4/ADR have been described elsewhere (5, 13) and were grown as
floating cells in RPMI medium (Flow Laboratories) with 7.5% fetal
including 5 @M
nigericin and 2 @M
valinomycin.Under these conditions,
the intracellular pH approached the extracellular pH (18, 19), and a
linear relationship between fluorescenceratio and pH (within the range
calf serum.
6.8—7.5)was observed. In a control experiment, the pH was measured
The resistant
cells were cultured
in the presence
of doxo
rubicin (0.12 @M
for SW-l573/2R120 and 1.2 @M
for GLC4/ADR
cells) until 3—10
days before the experiments. All cells were screened
for Mycoplasma contamination by using the Mycoplasma T.C. rapid
detection
system
with
a 3H-labeled
DNA
probe
from
by a digitonin null-point technique adapted from Ref. 20. Both methods
gave similar results.
Gen-Probe
Inc.(San Diego, CA) before these series of experiments and were found
to be negative.
RNase Protection. RNase protection was carried out as described by
Bus et a!. (4). RNA samples (10 big)were hybridized with a 32P-labeled
human mdrl-specific probe, a 301-nucleotide mdrl complementary
DNA fragment, or with a mdr3-specific probe, a 301-nucleotide mdr3
complementary DNA fragment (obtained from Dr. J. Smit, The Neth
erlands Cancer Institute, Amsterdam). The protected fragment was
visualized by electrophoresis through an acrylamide/urea gel (19:1),
followedby autoradiography. A ‘y-actin
probe was included as a control
for RNA recovery.
Cellular
Drug Accumulation.
Initial
uptake
studies
were carried
RESULTS
Daunorubicin
Transport.
Initially,
we measured
daunorubicin
accumulation in SW-1573 and SW-l573/2R120 cells for 6 h,
and steady-state accumulation was reached within 60 mm (not
shown). Therefore, 60 mm was chosen for all further experi
ments. Compared to the parent cell lines, the steady-state
accumulation of daunorubicin was 70 and 90% decreased in
SW-1573/2R120 and GLC4/ADR cells, respectively (Fig. I).
To examine whether the plasma membrane formed a permea
bility barrier that actively kept daunorubicin
out
out of the cell, the
cells were permeabilized with digitonin after steady-state accu
as described before (15). Adherent cells in the logarithmic phase of
mulation had been reached (Fig. 1). Daunorubicin binding in
growth were trypsinized, washed, and resuspended in densities of 0.5—
parent cell lines was not significantly affected by this procedure.
3 x 10@cells/ml in Dulbecco's modified essential medium containing
This can be explained as follows: when daunorubicin accumu
20 mr@iHEPES (pH 7.3—7.4),10% heat-inactivated fetal calf serum,
lation is determined by passive diffusion, no concentration
and 0.025% DNase I (medium A). Cells were incubated at 37C,
radiolabeled drugs were added, and at intervals thereafter, 0.2-mi au
gradient
quota were removed and dispensed in 1.5 ml of ice-coldPBS. After two
cold washes, the cells were transferred to liquid scintillation fluid (Opti
present when steady state is reached, and therefore, permeabil
ization of the plasma membrane is without effect. However,
exposure of the resistant cells to digitonin led to a rapid influx
of daunorubicin and binding of daunorubicin to DNA (Fig. 1).
Thus, under steady-state conditions, a concentration gradient
of daunorubicin over the plasma membrane was present in the
resistant cells. Differences in pH over the plasma membrane
could not be responsible for the increase in daunorubicin bind
Phase III; LKB, Bromma, Sweden), and radioactivity was measured.
Values were corrected for amount of cell-associated radioactivity at
time zero at 0CC. Steady-state accumulation of drugs was measured in
a similar way as described before (16). After steady-state accumulation
ofdaunorubicin
had been reached,
in some experiments
20 @iM
digitonin
(DNase I was then not included) was added 5 mm before the end of the
drug accumulation
period
in order to permeabilize
the plasma
mem
brane. This method has been adapted from Ref. 17 and will be described
ing after permeabilization
in detail elsewhere.4
an extracellular
No daunorubicin
was lost from digitonin-permea
bilized cells during the washing procedures.
metabolism
was inhibited
by incubation
of cells in medium
A without
kit obtained from Sigma was used. Within 15 mm, the ATP concentra
tion decreased to 10—15%of the initial concentration. A further de
crease to about 5% was measured during the following 90 mm. Cells
@
a
U
(0
w
wereincubated for 15mm at 3TC prior to addition ofdrugs, to decrease
cellular ATP concentrations —90%.Viability of the cells as measured
with trypan blue was always
M.
Versantvoort,
H.
J.
N.
Feller,
H.
Dekker,
rfi
200
z
0
2a
ioo
E
a
from time points 0—5mm for the
Broxterman,
400
0.
resistant cell lines and 0—10mm for the sensitive cell lines.
H.
pH (Table 2). This small
300
intervals thereafter, drug effiux was stopped with ice-cold PBS, and
cellular retention of radiolabeled drugs was measured. Half-lives of
4 C.
were done at
pH of 7.35 ±0.05 (mean ±SD) which is
E
@94%during the incubation periods.
effiux were calculated
since the experiments
will be
0
Cells (0.5—1
x i0@cells/ml) were loaded for 30 mm with radiolabeled
drugs in medium C to obtain drug levelsin resistant cells similar to the
levels in the sensitive cells. Cells were washed with ice-cold PBS and
resuspended (0.2—0.5
x l0@cells/ml) in drug-free medium at 37T. At
daunorubicin
membrane
difference in pH could only have resulted in a small efflux of
daunorubicin during permeabilization ofthe plasma membrane.
Figs. 2A and 3A show the time course of[i4-'4Cldaunorubicin
accumulation in parental SW-l573 and GLC4 cells compared
glucose but containing 10 mM sodium azide and I mg/ml 2-deoxy-dglucose (medium C). In this medium cellular ATP concentrations
decreased very rapidly as measured by the decrease of luminescence
generated by the luciferin/luciferase reaction. The bioluminescent assay
@
over the plasma
slightly higher than the intracellular
To study the ATP dependence of drug transport, cellular energy
@
of daunorubicin
0
SW- 1573
SW-I 573/2R1 20
GLC4
GLC4/A0R
Fig. I. Daunorubicin binding in permeabilized SW-l573 cells. After steady
state accumulation of 0.5 @iM
[14-'4Cjdaunorubicin had been reached (), cells
C.
werepermeabilizedby addition of 20 @iM
digitonin (0) and incubatedfor another
5 mm. Error bar, SD of at least three independentexperiments.
M.
Kuiper, and J. Lankelma, Int. J. Cancer, in press.
18
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1992 American Association for Cancer Research.
REDUCED DRUG ACCUMULATION NOT MEDIATED BY Paj
Measurements of Intracellular pH. Daunorubicin is a weak
base and is passively transported over the plasma membrane
mainly in the uncharged form (21); therefore, transport of
daunorubicin will be highly influenced by a pH gradient over
the plasma membrane. To be able to estimate putative contri
butions of pH differences to the measured differences in dau
norubicin accumulation, we determined the intracellular pH of
parental and variant cells under the same conditions as used
for drug accumulation experiments. No significant differences
in intracellular pH could be measured between sensitive and
resistant cells under such conditions (Table 2). Energy synthesis
inhibitors, sodium azide and deoxyglucose, however, caused a
rapid decrease in intracellular pH of @‘0.2—O.3
units in all cell
lines. In order to find out whether the increase in influx rate of
daunorubicin in medium C (Fig. 2B) could be caused (in part)
by this decrease in intracellular pH, the effect of nigericin on
initial uptake ofdaunorubicin was measured. In low K medium
nigericin enables electroneutral exchange of intracellular K@
ions for extracellular H' ions (18). Nigericin lowered the intra
a
U
(0
w
0
0
E
0.
a:
z
0
C
0
a
E
U
U
a
0
10
20
30
time in minutes
a
a
U
(0
w
0
100
80
0
E
0.
60
a:
z
40
0
C
0
a
a
20
U
0
a
500
a
0
(0
w
0
E
0
0
30 60 90 120
400
0
E
0. 300
time inseconds
z
Fig. 2. Energy-dependence of daunorubicin (DNR) accumulation in SW-1573
cells. 5W-1573
(0, •)and SW-1573/2R120
200
0
(Lx, A) cells were exposed to 1 MM
C
0
114-'4Cldaunorubicinunder control conditions (mediumA, pH 7.3 (0, E@)l
or in
medium C, pH 7.3 (A, •)and sampled at various intervalsfrom 0—30
mm (A).
Point,mean(bar,±SD)
ofat leastthreeexperiments.
A representative
experiment
a
100
E
for the uptake during the first 2 mm is shown in B.
0
0
a
a
i
and SW-i573/2Ri20
30
60
0
E
I 20
a:
3B). The initial rates of daunorubicin uptake calculated from
SW-i573
20
(0
w
0
80
a
the linear curve are shown in Table I. The rate of initial
daunorubicin uptake was lower in the resistant cells than in
their parental cell lines. As a result of cellular ATP depletion,
the rate of initial daunorubicin uptake was increased by a factor
1.3 in the parent cell lines and a factor 2.5—3in the resistant
sublines. The effects of sodium azide and deoxyglucose on the
initial uptake suggest the presence of an energy-dependent
efflux component that manifests its effect at a very early stage
of the uptake.
Efflux of 1i4-'4Cldaunorubicin from drug-loaded cells was
slower from cells resuspended in glucose-free medium contain
ing sodium azide and deoxyglucose than in control medium
(Figs. 4, A and B). In control medium, daunorubicin was
extruded faster from GLC4/ADR cells (t½3 ±0.4 mm) than
from the GLC4 cells (t½15 ±5 mm). No detectable difference
in initial t,, between
10
time in minutes
to their variant sublines SW-i 573/2R1 20 and GLC4/ADR,
respectively. Cellular ATP depletion resulted in an increase of
the daunorubicin accumulation in resistant and sensitive cells.
However, the effects were most pronounced in the resistant
SW-l573/2Rl20
(2.5-fold) and GLC4/ADR (10-fold) cells.
Under energy-deprived conditions, the time course of dauno
rubicin accumulation was similar in GLC4 and GLC4/ADR
cells.
The initial uptake of daunorubicin was linear with time for
the first 2 mm in both sensitive and MDR cells (Figs. 2B and
@
0
30
60
90 120
time in seconds
Fig. 3. Energy-dependent daunorubicin accumulation (DNR) in GLC4 cells.
Time courses for 1 MM114-'4Cldaunorubicin accumulation in GLC4 (0, •)and
GLC4/ADR (Li,A) cellsweremeasuredunder the conditionsas describedin Fig.
2 with the modification that the pH ofmedium A and C was 7.4. In B, the uptake
of daunorubicin
during the first 2 mm is shown. Point, mean of at least two
independentexperiments,each performedin triplicate;error bar, SD.
Table 1 Rates ofinitial uptakeofdaunorubicin in parent and resistantcells
Cellswereexposedto I MM1I4-―Cldaunorubicin.
The uptake during the first
2 mm of exposure to daunorubicin was used to calculate the rate of initial uptake.
Data are the means ±SD of 2-6 (numbers inexperiments.Initial
parentheses)
uptake rate
cells/mm)Medium
(pmol/l0'
CSW-1573
A
SW-1573/2Rl20
GLC4
cells was
found, while less daunorubicin was retained in the SW-1573/
2R120 cells after 30 mm efflux (significant, P < 0.05, Student's
GLC4/ADR
a Statistical
t test).
different
39 ±9 (4)d
11 ±2 (6)
19±4(3)
30 ±3 (3)@
25±5(3)
7 ±2 (2)
(P
<
0.05)
Medium
30 ±4 (6)
compared
to control
21 ±2 (2)
in medium
A (Student's
t test).
19
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1992 American Association for Cancer Research.
REDUCED DRUG ACCUMULATION NOT MEDIATED BY Pgp
cells than in the wild-type SW-i573 cells. We also measured
VP-i6 and vincristine efflux after loading energy-deprived cells
for 30 mm with 10 @tM
[3H]VP-16 or 1 @M
I3Hlvincristine. The
loss of VP-16 from cells resuspended in control medium A
occurred very rapidly with equal efflux rates in parent (t½1.7
±0.2 mm) and resistant cells (t½2.0 ±0.4) mm. However, the
VP-i6 efflux under ATP-depleted conditions was 2-fold slower
for SW-1573/2R120 cells (1½
4.3 ±0.5 mm) than for SW-1573
cells (1½
2.0 ±0.2 mm). Thus, if transport of drugs under ATP
depleted conditions can be considered as a passive process (21),
the passive transport (in and out) of VP-i6 across the mem
brane is 2-fold slower in the resistant cells. Because VP-i6 was
extruded with equal rates from resistant and sensitive cells in
normal medium, an additional energy-dependent transport
mechanism should be present in the SW-1573/2R120 cells. For
vincristine, the efflux was slow with no more than 10% efflux
during the first 30 mm.
sw-I 573
C
U
80
0
C
a
60
C
0
C
a
a
40
a
a
C
a
0
20
a
0
0.
0
10
20
30
time in minutes
C
U
0
C
GLC4
80
a
C
0
Detection
80
Y
C
a
a
a
a
C
a
0
a
0.
40
20
__v.
0
0
10
20
30
40
50
80
time in minutes
Fig. 4. Effiux of daunorubicin from SW-1573 (A) and GLC4 (B) cells. Cells
ofSW-1573(O, •)andSW-l573/2R120(E@, A)and GLC4 (0, t) and GLC4ADR
(V, Y) were loaded for 30 mm with 1 MM114-'4Cldaunorobicin in medium C.
Release ofdaunorubicin was measured after suspending the cells in daunorubicin
free medium A (0, t@,0, V) or in medium C (•,
A, , Y). Error bar, SD of at
least three determinations.
fluorescenceCells
Table 2 Intracellular pH measured by BCECF
37'Cin were incubated with 5 MMBCECF-acetomethylester for 30 mm at
BCECFfluorescence
medium A (pH 7.4). The intracellular pH was calculated from
means±
@
@
@
@
@
valuesas describedin “Materials
and Methods.―
Data are the
(3)SW-l573/2R120
(3)GLC4
(2)GLC4/ADR
A
6.92±0.11
7.00 ±0.09
7.29 ±0.02 (2)
7.08 ±0.02 (2)
7.30±0.02(2)
MDR
is associ
;@:
Medium
7.31 ±0.11 (5)
7.30 ±0.04 (5)
mRNA. In humans,
7.50
SD from 2—5
(numbers in parentheses)
experiments.Medium
CSW-l573
of Pgp/mdri
ated with the presence of Pgp, encoded by mdri gene (1).
Recently, Herweijer et a!. (22) suggested that the homologous
mdr3 gene encodes for a functional drug pump only in B-cell
lymphocytic leukemias. Previously, it was shown by immuno
staining with monoclonal antibodies JSB-i and C219 that the
MDR cell lines SW-i 573/2R1 20 and GLC4/ADR had no
increased levels of Pgp (4, 13). Neither was any mdrl mRNA
expression detectable in the SW-1573/2Ri20 cells by sensitive
RNase protection and polymerase chain reaction (3, 4). No
mRNA overexpression of the mdrl gene was detected in the
GLC4/ADR cells by using mRNA Northern blotting (13). In
order to assure the absence of Pgp in these cells, an RNase
protection assay with an mdri probe was performed (Fig. 6).
As a positive control for the sensitivity of this analysis, we used
the low-resistant KB8—5cells (23). Mdri mRNA expression
7.06±0.06
.I
7.20
0.
7.10
-t
0
A
,,
7.00
6.90
Os,@:
cellular pH @-0.4—0.5
units within 1 mm, while the initial pH
6.80
@:
.
.
was recovered within 3—5mm (Fig. 5). Nigericin, however, did
0
1
2
3
4
5
not affect the uptake of daunorubicin in SW-i573 and SW
1573/2R1 20 cells in the first 2—3mm when the pH was signif
time in minutes
icantly lowered (data not shown).
Fig. 5. Changesin intracellularpH (pHi) due to nigericin.Change of intracel
Transport of Vincristine and VP-16 in SW-1573 Cells. In the lular pH of SW-1573 (0) and SW-1573/2R120 (z@)in response to nigericin
addition was recorded by using the fluorescent dye BCECF. Data are from one
resistant SW-i573/2R120
cells, the accumulations of 1
pH
vincristine (3) and 10 @zM
VP-165 were reduced to 30—35% experiment(in duplicate),in whichcontrol pH ofSW-l573/2R120 was —0.1
unit lower than in SW-1573 cells.
compared to the drug accumulations in the SW-1573 cells. In
response to cellular ATP depletion, the accumulations of yin
Table 3 EffectofcellularATP depletionon accumulationofvincristineand VP
cristine and VP-i6 increased in the SW-i573/2R120
cells to a
greater extent than in the SW-i 573 cells (Table 3). In the wild
type as well as the resistant cells, the relative increase in
cellsInitial
16 in SW-1573
calculatedfrom
uptake rate of 10 MMVP-16 and 1 MMvincristine (VCR) was
mm(VCR).
the linear course of drug uptake during the first (VP-16) or first 2
andaccumulation
Data in ATP-depleted cells are percentages of initial uptake rate
accumulation of vincristine as a result of ATP depletion was
accompanied by a similar increase in initial uptake rate.
For VP-i6 no significant difference in steady-state accumu
lation between wild-type and resistant cells was measured under
cellular energy-depriving conditions, while under such condi
tions the initial uptake rate was still 2-fold lower in the resistant
eachperformed in medium A. Values are means of 3 experiments ±SD,
in triplicate. Data in ATP-depleted cells versus control were signifi
cantly different, P < 0.05, test.Initial
according to Student's t
accumulation(%)VCR
uptake rate (%)
VP-16SW-1573
201±75SW-l573/2R120
145±35
212±36
SD. R. A. Mans, G. P. van der Schans, E. G. E. de Vnes, S. de Jong, M. V.
M. Lafleur,J. Retèl,H. M. Pinedo, and J. Lankelma,manuscriptin preparation.
VP-l6
VCR
197±18
241±78
151±26
251±62
599±238
20
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REDUCED DRUG ACCUMULATION NOT MEDIATED BY Pgp
belonging to a superfamily ofATP-dependent transporters have
been identified in organisms from bacteria to eukaryotes (25,
26). Since Pgp seems to play a minor role in lung cancer (1 1),
we searched for yet unidentified drug transporters in these two
MDR lung tumor cell lines.
Previously, we found that, similar to Pgp MDR cells, the
SW-1573/2Ri20 non-Pgp MDR cells had a decrease in nuclear
anthracycline content compared to the parent cells (27). Assum
ing that the accumulation of daunorubicin is predominantly
determined by binding to DNA (28, 29), we reasoned that cells
with a reduced daunorubicin accumulation, caused by an active
outward transport of the drug, could be detected by means of
permeabilization of the plasma membrane (17, 29). Such an
approach to probe putative drug transporters is required, since
we are still lacking specific resistance modifiers for these non
Pgp-mediated MDR cells. We will document such a method
using a low concentration of digitonin in detail elsewhere.4
Here, we showed that the daunorubicin binding upon digitonin
exposure was increased about 2.5-fold in the SW-1573/2R120
and 12-fold in the GLC4/ADR cells but not in the parent cells.
Since daunorubicin is thought to be transported across the
membrane in its uncharged form (21), the cellular accumulation
of daunorubicin is highly dependent on a pH gradient across
the plasma membrane (21, 29). From equations reported by
Skovsgaard and Nissen (30), we calculated that the pH in the
SW-i573/2R120 cells should be 0.6 units higher than in the
parent cells, in order to explain the difference in daunorubicin
accumulation at steady state between these cells solely by a
change in intracellular pH. However, the intracellular pH,
determined under conditions of drug accumulation measure
ments, was the same in resistant and sensitive cells. The increase
of daunorubicin binding after permeabilization of the plasma
membrane, therefore, strongly suggests that the reduced dau
norubicin accumulation in the resistant cells is related to
changes of drug transport at the plasma membrane. Therefore,
the influx and efflux properties of daunorubicin transport in
SW-i 573/2R1 20 and GLC4/ADR cells were studied in more
detail.
In the resistant GLC4/ADR cells, the decrease in daunorub
CO
MOR1
@
ACT/N
“@
icin accumulation
No increased
expression
was detectable
in the sensitive
GLC4 or in the GLC4/ADR and the SW-i573/2Ri20
MDR
cell lines. Also, mdr3 mRNA was not overexpressed in the
MDR SW-i573/2Ri20
and GLC4/ADR cell lines, using
RNase protection analysis (data not shown). Moreover, the
absence of C2l9 staining proves the absence of mdr3 Pgp (24).
DISCUSSION
cells was ap
1573 cells. Thus, in the resistant
SW-1573/2R120
cells altera
tions in (apparent) influx rather than alterations in drug efflux
can account for the drug accumulation defects.
A lower influx of drugs in resistant cells with a Pgp-related
accumulation defect is not an uncommon feature of this phe
The characteristics ofdaunorubicin transport in two resistant
lung cancer cell lines, SW-i 573/2Ri 20 and GLC4/ADR, were
studied
initial drug efflux from preloaded
parent in SW-1573/2R120 compared to SW-1573 cells in
standard medium. The relatively low resistance to daunorubicin
of the SW-1573/2R120 cells (about a factor 4) might be a
reason that no increased daunorubicin efflux was apparent in
the SW-1573/2R120. However, in the Pgp-mediated MDR
KB8—5cells (23), which have low resistance for daunorubicin,
daunorubicin was extruded 2-fold faster from the resistant
KB8—5cells than from the parent KB3—1cells (not shown).
The lower steady-state accumulation in the resistant cells was
accompanied by a reduced initial uptake rate of daunorubicin.
The initial uptake rates of vincristine and VP-16 were also
decreased in the SW-1573/2R120 cells compared to the SW
was clearly detectable in the KB8—5cells, while no expression
in the parental KB3—1cells was found. Furthermore, a low level
of mdri mRNA expression was detected in the sensitive SW
1573 cells. No mdri
was mainly caused by the 5-fold faster dau
norubicin efflux. Such a large increase in efflux rate points to
a high capacity of active drug transport in GLC4/ADR cells.
Fig. 6. P-glycoprotein expression. Expression of the mdrl gene was measured
by an RNase protection assay (see “Materialsand Methods―).The positions of
the protected fragments for mdrl and -y-actin are indicated.
in detail because these cells have a reduced drug accu
mulation and display a multidrug cross-resistance pattern and
lack mdri mRNA expression. Therefore, MDR in these cells
seems to be based at least partly on other drug handling mech
anisms than Pgp. An increasing number of membrane proteins
notype. Sirotnak et aL (31) showed that the influx of vincristine
in MDR Chinese hamster cells was 24-fold lower compared to
the parental cells. Also, for other drugs, including doxorubicin
21
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REDUCED DRUG ACCUMULATION NOT MEDIATED BY Pgp
(17), daunorubicin
in MDR
(32), and VP-l6 (33, 34), a reduced influx
cells has been reported.
Thus, a reduced
drug accu
mulation caused by overexpression ofPgp might manifests itself
not only by an increase in drug efflux but also by a decrease in
apparent drug influx rate (17, 21, 33). Model calculations for
daunorubicin transport in Ehrlich ascites cells have shown that
the apparent uptake of daunorubicin can be influenced imme
diately after offering the drug to cells with an outwardly directed
drug pump with high affinity (Km 10_s M) (35). Blocking of this
pump would then result in an increase in apparent uptake of
daunorubicin. In the present experiments, the initial uptake of
daunorubicin increased as a result of cellular energy depletion.
However, we were unable to measure saturation of influx rates
in SW-i 573 and SW-i 573/2R1 20 cells, since the apparent
influx rate was linear with external daunorubicin concentrations
from 108 to 4 x iO_6 M in both cell lines (not shown). Thus, a
putative ATP-dependent drug efflux mechanism in these cells
would have a higher capacity for daunorubicin. An auternative
explanation for an apparent slower drug influx in Pgp MDR
cells was given by Raviv et aL(36). In this model, on its way to
the cytoplasm, a drug is bound to Pgp in the plasma membrane
and is immediately pumped out. Other putative drug transport
ers might manifest themselves in the same way by a decrease in
apparent drug influx rate.
Another indication for the presence of an active drug trans
porter can be obtained by studying the ATP dependence of drug
transport as was initially performed by Danøet aL (37). In both
non-Pgp-mediated MDR lung cancer cell lines, we found a
large increase in daunorubicin accumulation in response to
cellular ATP depletion. In the resistant cells, the increase in
daunorubicin was accompanied by a 2.5- to 3-fold faster uptake
of daunorubicin. In the GLC4/ADR cells, the enhanced dau
norubicin
extrusion
was inhibited
cells compared to the SW-i573 cells, pointing to possible
differences in energy-dependent drug transport mechanisms
between the two non-Pgp MDR cell lines. For three other
MDR cell lines without Pgp expression, a human leukemic cell
line, HL-60/ADR
(7), a human fibrosarcoma cell line,
HTiO8O/Dr4 (6), and a human large cell lung carcinoma cell
line, COR-L23/R (10), an enhanced efflux of doxorubicin and
daunorubicin has been reported. For HL-60/ADR cells, it has
been suggested that an Mr 190,000, ATP-binding membrane
protein was involved in efflux of the drugs out of the cells (8).
Recently, we developed an IgG2b antibody (LRP56) against
SW-i 573/2R120 cells (38). The antibody showed intense stain
ing of the non-Pgp MDR SW-i 573/2R1 20 and the GLC4/
ADR cells during acetone fixation, while the parental cell lines
SW-1573 and GLC4 stained only weakly. We speculate that
the protein recognized by this antibody is involved in the
mechanisms
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Energy-dependent Processes Involved in Reduced Drug
Accumulation in Multidrug-resistant Human Lung Cancer Cell
Lines without P-Glycoprotein Expression
Carolina H. M. Versantvoort, Henricus J. Broxterman, Herbert M. Pinedo, et al.
Cancer Res 1992;52:17-23.
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