Download Plasma Membrane Lipid Structural Order in Doxorubicin

[CANCER RESEARCH 43, 5533-5537,
November 1983]
Plasma Membrane Lipid Structural Order in Doxorubicin-sensitive
and -resistant P388 Cells
A. Ramu,1 0. Glaubiger, I. T. Magrath, and A. Joshi
Department oÃ-Radiation and Clinical Oncology, Hadassah University Hospital, Jerusalem, Israel [A.R.]; Pediatrie Oncology Branch, National Cancer Institute, Bethesda,
Maryland 20505 ¡O.G.,U.M.]; and Organic and Biological Chemistry Branch, Food and Drug Administration, Washington, D. C. 20204 [A.J.]
ABSTRACT
We have studied the structural order of the lipid phase of
plasma membranes from P388 murine leukemia cells and from
a Doxorubicin-resistant subline, P388/ADR, using electron spin
resonance spectroscopy and fluorescence depolarization mea
surements. Measurements of the order parameter, S, following
incubation of cells from both lines with the A/-oxyl-4'-4'-dimethyloxazolidine derivative
for the resistant cells at
(4-37°). Fluorescence
incubation of the cells,
of 5-ketostearic acid show higher values
all temperatures where S was measured
depolarization measurements following
or cell fractions, with 1,6-diphenylhexa-
triene indicate more restricted motion of the probe in resistant
cells. These measurements also show increased amounts of
cytoplasmic lipid in the resistant cells.
The higher degree of structural order in the lipid phase of the
plasma membranes of P388/ADR cells and their larger intracellular lipid content may account for the decreased rate of intracellular accumulation of anthracycline drugs (and other com
pounds) seen in these cells and, in part, for their relative resis
tance to the cytotoxic effects of these drugs.
INTRODUCTION
A number of ADR2-resistant cell lines have been developed by
repeatedly exposing sensitive cells to low doses of drug in vivo.
(6, 21, 32). The resistant sublines also show resistance to other
related anthracyclines (daunorubicin, cinerubin A, carminomycin,
and nogalamycin), as well as to drugs which have chemical
structures unrelated to anthracyclines, including vincristine, vinblastine, mithramycin, ellipticine, actinomycin D, and emetime (6,
7, 14, 17, 18, 20, 21, 32). Cell lines which have been made
resistant to other drugs, chemically unrelated to anthracyclines,
can also demonstrate cross-resistance to the anthracyclines as
well. Examples include cells which have developed resistance to
maytansine, vincristine, vinblastine, actinomycin D, and terephthalanilide (1, 2, 7, 8, 22, 23, 40, 47).
Several studies have indicated that intracellular drug accu
mulation is decreased in resistant lines, both for the drug used
to prepare the resistant subline and for drugs to which the
subline is cross-resistant. (2, 4, 8, 17, 18, 28, 29, 32, 39, 40). It
is not clear whether this decreased drug accumulation is the
result of decreased drug influx, increased drug efflux, or both.
The mechanism of drug influx into these cells is uncertain. ADR
may enter the cell by diffusion or by a membrane-associated
carrier transport system (16, 17, 37, 38). In either case, the rate
of drug uptake would be expected to depend, in part, on the
1To whom requests for reprints should be addressed.
2 The abbreviations used are: ADR, Doxorubicin; DPH, 1,6-diphenylhexatriene;
PBS, phosphate-buffered saline; ESR, electron spin resonance; 5NSA, W-oxyl4',4'-dimethyloxazolidine
derivation of 5-stearic acid.
Received August 16, 1982; accepted July 13, 1983.
NOVEMBER
1983
ease of molecular motion in the lipid phase of the membrane,
and inversely on the lipid packing density.
Techniques have now been described which permit more
detailed investigations of lipid structural order in membranes (10,
13, 15, 33-35, 47). In the present study, we have used electron
paramagnetic resonance spectroscopy and spin-labeled stearic
acid probes as well as fluorescence depolarization measure
ments (using DPH as a reporter molecule), to examine the degree
of structural order of the lipid domains of plasma membranes of
P388 cells and of a subline resistant to anthracyclines (P388/
ADR). We attempted to obtain measurements on whole cells,
rather than subcellular fractions, in order to minimize the difficul
ties in making sufficiently pure and structurally unaltered mem
brane fractions.
MATERIALS
AND
METHODS
Cell Culture and Membrane Preparation. Mouse P388 leukemia cells
and a subline resistant to ADR [P388/ADR, described previously by
Johnson ef al. (21 )] were grown in suspension culture in Roswell Park
Memorial Institute 1640 Medium (Grand Island Biological Co., Grand
Island, N. Y.) supplemented with 10% heat-inactivated fetal bovine serum
(Grand Island Biological Co.), 10 MM2-mercaptoethanol (Sigma Chemical
Co., St. Louis, Mo.), penicillin base (50 units/ml), and streptomycin base
(50 Mg/ml) (both from Grand Island Biological Co.,). Cell densities were
measured using a Coulter Counter (ZB1; Coulter Electronics Ltd., Harpenden, Herts, England). Cells were transferred to fresh medium every
4 days to sustain exponential growth. Initial cell densities were 10s cells/
ml and, after 4 days, their density reached 1 to 2 x 106 cells/ml. In all
studies where P388 and P388/ADR cell characteristics were compared,
measurements were performed with cells harvested on the fourth day of
growth. With every 5 to 10 transfers, the effect of 1 x 10~7 M ADR on
the growth rate of both cell lines was measured to verify the consistency
of the differential sensitivity to ADR.
Membrane Preparations. Membranes were isolated by the method
used in our laboratory in the past for malignant cells, as described
previously (42). In brief, 1.5 x 109 cells of both lines were washed 3
times with Dulbecco's PBS (9), then resuspended in hydroxyethylpiperazine-AT-2-ethanesulfonic
acid buffer (pH 7.4) containing 140 mw NaCI
and 1.0 HIM MgCI2. The cells were then disrupted by nitrogen cavitation
(500 \j>\20 min). After centrifugation at 450 x g for 5 min, the supernatant
was centrifugea at 25,000 x g for 20 min, and the mitochondrial and
lysosmal pellets were discarded. The supernatant was centrifuged at
200,000 x g for 60 min, and the microsomal pellet was suspended on
0.1 M hydroxyethylpiperazine-W-2-ethanesulfonic
acid buffer (pH 7.4).
The suspension was then layered on a discontinuous sucrose gradient
(35%/45%) and centrifuged at 200,000 x g for 2 hr. The plasma
membrane band was collected at the upper interface. Electron micros
copy of this membrane band revealed large sheets and vesicles of various
sizes without other subcellular contaminants. No difference was ob
served between membranes obtained from ADR-sensitive or -resistant
P388 cells. As reported by us in another communication,3 no differences
3A. Ramu, D. Glaubiger, and H. Weintraub. Differences in lipid composition of
Adriamycin sensitive and resistant P388 cells, submitted for publication.
5533
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A. Ramu et al.
Table 1
Temperaturedependence of the order parameter, S, in Adriamycin-sensitiveand -resistant P388 cells
For experimental details, see 'Materials and Methods
Magnitude of S
CeteP388
±0.002a
±0.006
±0.003
±0.001
±0.002
±0.006
0.724 ±0.00120°0.685
0.717 ±0.00825°0.668
0.677 ±0.00530°0.623
P388/ADR4°0.794 0.797 ±0.00315°0.708
0.637 ±0.00237°0.594
0.612 ±0.002
1Mean ±S.D.
were found between these cell lines in the total and plasma membrane
content of cholesterol, phospholipid phosphate, or protein. Triglycérides
were not detected in plasma membrane preparations. There were signif
icant differences in membrane-associated enzyme activities.4 However,
the activity of 5'-nucleotidase
and Na-K-ATPase (calculated per mg
protein) of the membrane preparations was enriched 15- to 20-fold
relative to the whole-cell lysate for both cell lines, with no significant
change in the relative specific activity.
ESR Measurements. From each line, 10' cells were washed 3 times
with PBS and then incubated in Hanks' balanced salt solution with 5NSA
(2.5 x 10~5 M; Syva Corp., Palo Alto, Calif.) in a volume of 100 p\ at 4°.
Samples were then transferred to a disposable capillary tube sealed at
one end and, after equilibration at a selected temperature, electron
paramagnetic resonance spectra were obtained using a Varian EPR E-9
spectrometer equipped with a temperature control accessory (Varian
Associates, Palo Alto, Calif.). The order parameter (13) was calculated
according to:
_
TÃ--T!
-C
-r; + 2r,;+ 2Cx1J23
where TÃ-and T! (in gauss units) are equal to one-half the separation of
the outer and inner spectral extrema, respectively,
minus 0.053 (TÃ-- Ti').
and C is 1.4 gauss
Under these experimental conditions, the signal of free 5NSA was
negligible compared to that of 5NSA bound to cells and did not interfere
with the spectral measurements. The reproducibility of the measure
ments was ±0.005 (S.D.) gauss.
Fluorescence Depolarization Measurements. Steady-state fluores
cence depolarization measurements of whole cells and isolated plasma
membranes were carried out according to the method described by
Shinitsky and Inbar (35, 36); 107 washed cells or their isolated mem
branes were exposed to DPH (Aldrich Chemical Co., Milwaukee, Wis.)
by incubation with 2 x 10~6 M DPH at 25°for 30 min. Measurements
were made at 25°with an Elscint Microviscosimeter
MV-1 (Elscint, Ltd.,
Haifa, Israel). Serial dilutions were performed in order to derive accurate
readings independent of light-scattering effects (28). The degree of
fluorescence polarization
according to:
(P) is internally computed
by the instrument
P = (/,, - /,)/(/,, + /,)
where I»and I, are the emission intensities in channels parallel and
perpendicular to the plane of polarization of the exciting light. The
reproducibility of the measurements was ±0.005 P units.
DPH Binding. From each line, 10e washed cells were incubated with
16 ßMDPH in PBS for 30 min at 25°, then washed 3 times with PBS,
and lysed with Triton X-100 (final concentration, 0.5%). DPN fluorescence
was then measured using an Aminco-Bowman spectrophotofluorometer
(American Instrument Co., Silver Spring, Md.). The samples were excited
at 365 nm, and emission measured at 418 nm. The amount of DPH
bound to the cells was calculated from DPH standard curves which were
linear in the 2 x 10"7 to 1 x 10~6 M concentration range.
Cytological Studies. Cells from each line were spun for 5 min in a
cytocentrifuge (Shandon Southern Instruments, Inc., Sewickley, Pa.),
and the slides were immediately stained with oil red O.
RESULTS
Spin-Label ESR Measurements.
Cultured cells of each line
(transfers 30, 31, and 43) were labeled with 5NSA, and spectra
were measured at 4-37° as described in "Materials and Meth
ods." As shown in Table 1, in each cell line, the magnitude of the
calculated order parameter S was inversely related to tempera
ture. At each temperature, the S value of P388/ADR cells was
higher than the S value for P388 cells.
Whole-Cell DPH Fluorescence Polarization Measurements.
The P values obtained for DPH-labeled cells showed remarkable
stability (Table 2). The P value obtained for P388/ADR cells was
consistently lower than that of P388 cells. The whole-cell P value
was found to depend on the length of the incubation time with
DPH (Chart 1). In both cell lines, P declined with time. The rate
of this decline was more rapid in P388/ADR cells, and steadystate values were considerably lower than those measured for
P388 cells. Accurate measurements could not be obtained for
incubation times shorter than 2 min, because the total cell DPH
fluorescence was too low.
DPH Binding. The amounts of DPH bound to 108 cells of each
line incubated with 16 /ÕMDPH at 25°for 30 min were compared.
It was found that P388/ADR cells bind 40 ±5% more DPH than
do P388 cells.
DPH Fluorescence Polarization of Cell Fractions. Cell frac
tions obtained in the process of isolating plasma membranes
were labeled with DPH, and the P value of each fraction was
determined. Results are presented in Table 3. For each cell line,
the cell homogenate, obtained after nitrogen cavitation, had a
measured P value which was close to that obtained for whole
cells. Similar results were obtained for the pelleted nuclei (450 x
g; 5-min pellet). Supernatants obtained by centrifuging the cytoplasmic fraction at high speed (200,000 x g; 60 min) exhibited
some turbidity near the top of the centrifuge tubes. This turbid
layer was more distinct in supernatants obtained from P388/
ADR cells. After incubation with DPH, this layer yielded P values
that were significantly lower than those obtained for whole cells.
The P value obtained for the turbid supernatant layer of P388/
ADR cells was lower than that measured for P388 cells.
Table 2
Whole-cellDPH fluorescence polarization P value of P388 and P388/ADRcells
For experimental details, see "Materials and Methods."
Cell
Fourth transfer
in culture
52nd transfer
in culture
P388
4A. Ramu, D. Glaubiger, P. Soprey, G. H. Reaman, and N. Feuerstein. Differ
ences in some plasma related activities between Adriamycin sensitive and resistant
P388 cells, submitted for publication.
5534
P388/ADR0.235
0.1870.237
Mean ±S.D. of measure
ments over 3 mos.
±0.009 (20)"
0.1920.239
0.195 ±0.012 (19)
" Numbers in parentheses, number of measurements.
CANCER
RESEARCH
VOL. 43
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Membrane Lipid Order in ADR-resistant Cells
The P values measured for plasma membrane bands were
also significantly different from those obtained for whole cells. In
both cell lines, the plasma membrane P value was higher than
that of whole cells. However, compared with P388 cells as well
as membranes of P388 cells, membranes of P388/ADR cells
have a higher P value.
Cytology. P388 and P388/ADR cells stainedwith Oil Red O
were photographed using a Lietz microscope with no filter. As
shown in Fig. 1, P388/ADR cell cytoplasm contains many more
droplets than that of P388 cells.
DISCUSSION
It has been shown previously that modulation of the packing
density of plasma membranes, by altering ratios of saturated to
unsaturated fatty acids, is reflected by changes in the order
parameter, S, calculated from ESR spectra of spin-labeled
probes incorporated into the membranes (3,10, 25). It has also
been shown that short-term (<30 min) incubation of cells with
fatty acids results in incorporation of >80% of the labeled ma
terial into the cell membrane (24, 41). Using similar conditions,
studies on the incorporation of 5NSA, the spin-labeled probe
used in the studies presented here, indicated that this probe was
also located in the cell membrane (12). Spin-labeled saturated
fatty acids, such as 5NSA, when they are incorporated into
plasma membranes, are thought to be oriented parallel to the
acyl chains of the membrane phospholipids (10,13,15). Based
.280r
.260
.240
o-
.220
••
••
o
P388
•»
••
«•
«
O
.200
.180
o o
.160
10
20
30
P388/ADR
oo
o o Oo
60
60
Time (mini
Chart 1. DPH fluorescence polarization values (P) as a function of time of
incubation for P388 (•)and P388/ADR (O) cells. Cells were incubated with 2 x
10~6 M DPH solution for the time shown (abscissa) and washed with PBS, and the
P value was then determined. Steady-state values are lower for P388/ADR cells
than for P388 cells, although the extrapolated zero-time P value is higher for P388/
ADR cells.
upon the considerations outlined above, measurements on whole
cells incubated with 5NSA are thought to primarily reflect the
order parameter of the cell membrane. In further support of this
view, measurements of order parameter made on isolated mem
branes of another murine leukemia cell line, L1210, which have
incorporated 5NSA, are similar to those reported here (3).
The other probe molecule used in the studies presented here,
DPH, has different characteristics. In structures such as cell
membranes, in which both lipid domains and hydrophilic regions
are present, DPH distributes primarily into the hydrophobic lipid
domains (27). The steady-state fluorescence polarization value,
P, for DPH, is thought to reflect the degree of structural order of
the lipid molecules surrounding the DPH probe (19, 26). In
contrast to 5NSA, which distributes primarily into cell membranes
when incubated with whole cells, DPH, a highly lipophilic mole
cule, distributes throughout intracellular lipid-containing struc
tures as well as into the cell plasma membrane (5, 30, 31).
Consistent with previous reports, we have shown in this study
that the P values of cytoplasmic cell fractions are lower than
those obtained for whole cells, while values measured for isolated
plasma membranes are higher (11, 31, 43, 46), since the wholecell P is a weighted average of DPH behavior in cytoplasm and
bution of the membrane-bound DPH to the whole-cell P value,
we measured the time dependence of P in whole cells. It can be
assumed that, when cells are incubated with DPH, the probe
binds to the plasma membrane prior to its binding to intracellular
structures. Therefore, we conclude, from the decline of P with
time until steady state is reached, that, as was shown for cell
fractions, the plasma membrane P value is higher than that of
intracellular structures. The lower steady-state P values of P388/
ADR cells, compared to those of P388 cells, are in accord with
the differences found in P values of their cytoplasmic turbid
fractions (Table 3). However, it may also reflect differences in
the quantity of intracellular lipids, as indicated by the observed
difference in the quantity of the turbid supernatant fractions
obtained from these cells lines, as we have reported previously.3
A difference in the amount intracellular lipid between these cell
lines is further supported by the following observations, (a)
Although no difference was noted in DPH fluorescence of plasma
membranes isolated from P388 or P388/ADR cells, the amount
of DPH bound to whole P388/ADR cells was significantly higher
than the amount bound to P388 cells, (b) Upon staining with oil
red O, we observed that P388/ADR cells contain a significantly
larger number of intracellular lipid vacuoles than do P388 cells
(Fig. 1).
It was not possible to determine plasma membrane P values
reliably from extrapolation of the time-dependent measurements
of whole-cell P values to zero time, because measurements
could not be made at short incubation times (<2 min). Measure
ments of DPH P values for isolated plasma membranes, how
ever, are consistent with the data obtained from ESR measure-
Table3
DPH fluorescence polarization P values of Adriamycin-sensitive
For experimental details, see "Materials and Methods."
CellP388
0.004'
P388/ADRCell
Mean ±S.D.
NOVEMBER
1983
nemogenate
(after nitrogen
cavitation)0.242
±0.004a
of 450
xpellet0.247
g, 5-min
±0.003
0.195 ±0.003Suspension
0.206 ±0.006Turbid
and -resistant P388 cell fractions
fraction of
200,000 x g 60-min
supernatant0.147
±0.002
membrane
band0.275
±0.004
0.1 33 ±0.005Plasma 0.293 ±
5535
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A. Ramu et al.
ments. Both indicate that the structural order in the lipid domains
of plasma membranes of P388/ADR cells is higher than in P388
cells.
This difference in structural order of the lipid portion of plasma
membranes between ADR-sensitive and -resistant cells may
explain the lower rate of accumulation of some chemically unre
lated compounds in the drug-resistant cells and the wide spec
trum of cross-resistance.
A similar correlation between drug uptake and membrane
fluidity has been shown by others for methotrexate (3). Recently,
it was suggested that the plasma membrane is the primary target
for the cytotoxic action of ADR (44,45). Therefore, an alternative
explanation for the difference in sensitivity of P388 and P388/
ADR to this drug is that resistance results from some difference
in the interaction of ADR with the surface membranes of these
cell lines, such as lower drug binding by the more rigid mem
brane. The increased content of intracellular lipid in P388/ADR
cells may serve as an intracellular storage reservoir for the drug,
diverting it from its site of cytotoxic action, and, thus, may also
contribute to the apparent relative resistance of these cells to
ADR.
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CANCER
RESEARCH
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VOL.
43
Membrane Lipid Order in ADR-resistant Cells
Fig. 1. Photomicrographs
ADR cells, x 1250.
NOVEMBER
1983
of P388 œlls (left) and P388/ADR cells (right) stained with oil red O. Number of ¡ntracellularlipid-containing structures is higher for P388/
5537
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Plasma Membrane Lipid Structural Order in
Doxorubicin-sensitive and -resistant P388 Cells
A. Ramu, D. Glaubiger, I. T. Magrath, et al.
Cancer Res 1983;43:5533-5537.
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