[CANCER RESEARCH 44, 4978-4980,
November 1984]
Involvement of Plasma Membrane Lipid Structural Order in Adriamycin
Resistance in Chinese Hamster Lung Cells1
DavidA. Rintoul2and MelvinS. Center
Division of Biology, Kansas State University, Manhattan, Kansas 66506
ABSTRACT
Plasma membrane preparations from Chinese hamster lung
cells, which are resistant to the antitumor agent Adriamycin,
were analyzed using fluorescence polarization of the membrane
lipid probe frans-parinaric acid. The results of these studies
reveal that membranes from several drug-resistant isolates have
a substantial decrease in lipid structural order relative to mem
branes from drug-sensitive cells. Additional studies have shown
that certain isolates are unstable and undergo a sequential
phenotypic reversion after continuous passage in culture. Thus,
we have identified cells which have reverted for membrane lipid
physical changes but which still remain highly resistant to Adri
amycin. At later passages, these cells are found to revert to drug
sensitivity. These results indicate that an alteration of plasma
membrane lipid structural order is not an essential component
of the Adriamycin-resistant phenotype. However, in certain iso
lates, drug resistance and changes in membrane physical prop
erties are both associated with an unstable genetic element.
INTRODUCTION
Previous studies have shown that cells resistant to the antitumor agent Adriamycin are highly defective in the cellular ac
cumulation of drug (2, 3, 5, 15, 16). This defect appears to be
primarily due to impaired drug transport into the cell (15, 16),
and/or a major enhancement of a drug efflux mechanism (4, 5,
16). The results of these studies suggest that plasma membrane
alterations may make a significant contribution to the drugresistant phenotype. Consistent with this is the finding that
plasma membranes of drug-resistant cells contain a phosphorylated glycoprotein (P-180) which is not detected in cells sensitive
to Adriamycin (1,2). Recent studies have provided evidence that
there is a strong correlation between the presence of this protein
and drug resistance (1). Additional studies also indicate that
phosphorylation plays an important role in regulating the biolog
ical activity of P-180 (1, 2).
Recently, several laboratories have reported that cells resist
ant to Adriamycin have an alteration in the lipid "fluidity" of the
cell surface (9,13,18).3 It was thus speculated that drug resist
ance may be related to this cellular change. In the present study
1This investigation was supported in part by Research Grant CA-28120 from
the National Cancer Institute, Department of Health and Human Services, and by
American Cancer Society Grant IN-115, through the Mid American Cancer Center.
This study is Contribution 82-437-j from the Kansas Agricultural Experiment Station,
Division of Biology, Kansas State University, Manhattan, KS 66506.
2To whom requests for reprints should be addressed.
3 In the absence of independent measurements of fluorescence lifetimes, de
creases in steady state fluorescence depolarization cannot be unambiguously
attributed to increases in "fluidity." Thus the expression fluidity, or decrease in lipid
structural order, is used here in its broadest sense, denoting changes in the rate
of rotation and/or the distribution of the fluorophore in the anisotropic lipid bilayer
of plasma membranes.
Received December 6,1983; accepted July 30,1984.
4978
we use steady-state fluorescence polarization to determine that
membrane physical properties of resistant cells are significantly
altered relative to membranes from cells sensitive to drug. How
ever, by analyzing cells which have reverted to drug sensitivity,
we show that the alterations in these membrane physical prop
erties are not required in order for a cell to exhibit drug resistance.
MATERIALS AND METHODS
Cells. Chinese hamster lung cells resistant to Adriamycin were isolated
as described previously (3). Both sensitive and resistant cells were
cultured in Dulbecco's modified Eagle's medium (Grand Island Biological
Co., Grand Island, NY) supplemented with 10% fetal calf serum.
Plasma Membranes. Plasma membranes from sensitive and resistant
cells were isolated as previously described (10). The crude cell membrane
preparation was applied to a discontinuous sucrose gradient (15 to 60%
sucrose) and centrifugea for 2 hr at 35,000 rpm in the Spinco SW50.1
rotor. The plasma membranes and endoplasmic reticulum fractions were
collected, diluted in 0.01 M Tris-HCI (pH 7.6), and thereafter pelleted by
centrifugation for 1 hr at 35,000 rpm in the Spinco SW50.1 rotor. The
isolated membrane preparations were suspended in 0.01 M Tris-HCI (pH
7.6). The purity of the plasma membrane fraction was determined as
described previously (2). As determined by electron microscopy and
marker enzyme analysis (12), there is no detectable difference in the
plasma membranes from drug-sensitive and -resistant cells.
Fluorescence Polarization. Fluorescence polarization analysis, using
TPNA4 (10, 11) (obtained from Dr. R. D. Simoni, Biological Sciences,
Stanford University, Stanford, CA) was performed as previously de
scribed (11). Corrections for scattering depolarization were made, when
necessary, by the method of Lentz ef al. (7). Computer analysis and
curve-smoothing methods have also been previously reported (11).
RESULTS
The results of a typical fluorescence polarization experiment,
using TPNA, are shown in Chart 1. Chart "\A shows the temper
ature-dependent fluorescence polarization of the probe in plasma
membrane preparations from sensitive and resistant cells. Stand
ard deviations of the values shown are not presented in this
figure, since in all cases the deviations are less than 0.03 unit. It
is apparent that the polarization ratio, which is inversely corre
lated with probe rotational mobility (14), was lower in the resistant
cell membranes. This difference was maintained throughout the
temperature range of 10-45°. Equivalent preparations from a
separate resistant isolate also exhibit a decreased polarization
ratio throughout this temperature range (Chart 1). As shown in
Table 1, the plasma membranes from 3 independent isolates of
Adriamycin-resistant cells exhibit similar changes in fluorescence
polarization using TPNA as a probe of lipid motion. These results
suggest that plasma membranes from resistant cells exhibit less
structural order than do those isolated from cells sensitive to
'The abbreviations used are: TPNA, frans-parinaric acid (all-trans-9,11,13,15octadecatetraenoic
acid); DMS, double minute chromosomal spheres.
CANCER RESEARCH
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VOL. 44
Membrane Changes in Adriamycin-resistant
Chinese Hamster Lung Cells
2.1
2.0
2.0
1.9
1.9
1.8
•1.8
1.7
•1.7
1.6
-1.6
1.5
•1.5
1.4
-1.4
2.0
1.9
1.8
1.8
1.5
1.4
10
20
30 40 50
TEMPERATURE
10
20 30
(°)
40
50
Chart 1. Fluorescence polarization of TPNA in plasma membranes and endoplasmic reticulum from sensitive and resistant Chinese hamster lung cells. Fluores
cence polarization determinations with TPNA were carried out as described in Ref.
11. Samples were cooled at 0.75°/min. Results are plotted as polarization ratios,
where /M and /. refer, respectively, to intensities emitted parallel and perpendicular
to the vertically polarized excitation. A and B, respectively, show fluorescence
polarization versus temperature in plasma membranes and endoplasmic reticulum.
O, HT-1 (sensitive) cell membranes; •,
ADR6 (resistant) cell membranes; x, ADR24
(resistant) cell membranes.
Table 1
Properties of Adriamycin-resistant
P*
Isolates
Drug uptake"
Degree of
resistance6
10
20
TEMP
30
40
60
Chart 2. Fluorescence polarization of TPNA in plasma membranes from drugresistant Isolate R3. Plasma membranes from Isolate R3 after passages 40, 70,
and 110 in culture were analyzed by fluorescence polarization; O, HT-1 (sensitive)
membranes; D, R3P40; *, R3P70; »,R3P110.
these cells were no longer less ordered than those from drugsensitive cells (Chart 2). These cells are however still highly
resistant to Adriamydn (Table 1). If R3P70 is grown until passage
110, we now find that these cells have reverted to drug sensitivity
(Table 1), and have the same membrane physical properties as
R3P70 (Chart 2).
(sensitive)R6R24R3P10R3P40R3P70R3P1101.001.121.121.121.100.950.981.00.200.250.250.250.301.01.02001701801801802.0
HT-1
DISCUSSION
P (37e) - /i .//.(sensitive)
/i i/U resistant)
Drug uptake was determined as described previously (3). Values are relative
to drug uptake in HT-1 cells.
c Relative concentration of drug that inhibits cell growth by 50%.
Adriamycin. Of interest is the finding that the TPNA fluorescence
polarization in endoplasmic reticulum was not altered in resistant
cells (Chart 1S), thus suggesting that this change in lipid physical
properties is specific for the plasma membrane.
In order to examine the involvement of membrane structural
order in Adriamycin resistance, selected revenants were isolated
and characterized. Isolation of these revenants is based on the
finding that Isolate R3 becomes sensitive to Adriamycin after
several passages in culture. We have found that by following
phenotypic reversion after several passages we can demonstrate
that distinct genetic loci are involved in membrane changes and
drug resistance. In these studies, we analyzed plasma membrane
lipid physical properties and drug resistance in the R3 isolate
after passages 10, 40, 70, and 110. As shown in Chart 2, the
TPNA fluorescence polarization in plasma membranes from
R3P40 is considerably lower than TPNA fluorescence polariza
tion in isolated plasma membranes from sensitive cells. This
isolate is also highly resistant to Adriamycin (Table 1). The early
passage R3 isolate, R3P10, also exhibits membrane physical
properties which are identical to those of R3P40 (Table 1). This
isolate is also highly resistant to Adriamycin (Table 1). However,
an analysis of R3P70 showed that the plasma membranes from
NOVEMBER
1984
In the present study fluorescence polarization has been used
to analyze the lipid fluidity of plasma membranes from cells
resistant to Adriamycin. Isolated plasma membranes were ex
amined, since studies with whole cells have been shown to be
unreliable, due to probe accumulation in ¡ntracellularlipid droplets
which are very fluid (8, 17). The results of the present study
demonstrate that membranes from several independent drugresistant isolates are considerably less ordered than are mem
branes from sensitive cells. This alteration in membrane physical
properties appears to be confined to the plasma membrane,
since similar changes are not observed for isolated endoplasmic
reticulum. Recently, several laboratories have also reported
plasma membrane fluidity changes in cells resistant to Adriamy
cin. Siegfried ef al. (13) have utilized electron spin resonance
spectroscopy to analyze fluidity changes in drug-resistant Sar
coma 180 cells. The results of these studies demonstrated that
several different drug-resistant isolates had an increase in mem
brane fluidity. Wheeler ef al. (18) have also found that the murine
tumor line, MDAY-K2, selected for Adriamycin resistance, ex
hibits an increase in membrane fluidity, as determined by fluo
rescence polarization analysis in the presence of diphenylhexatriene. In contrast to these results and those which we have
reported, Ramu ef al. (9) have found that Adriamycin-resistant
P388 murine leukemia cells exhibit a decrease in lipid fluidity, as
compared to drug-sensitive cells. These results were obtained
by analyzing fluorescence polarization of diphenylhexatriene in
cubated with cells sensitive and resistant to drug. Since all of
these studies were carried out without an analysis of cell reven
ants, it becomes of considerable interest to determine if an
alteration in membrane lipid physical properties is actually re
quired for a cell to exhibit drug resistance. The results of the
present study indicate that this is not the case. An analysis of
4979
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1984 American Association for Cancer Research.
D. A. Rintoul and M. S. Center
selected revertants reveals that cells can exhibit membrane
physical properties closely similar to the parent cell, but still be
resistant to Adriamycin. It should be pointed out that the changes
observed in fluorescence polarization values for the membranes
used in this study, and for the whole cells used in the previous
studies (9, 18), are not necessarily due solely to changes in
membrane lipid rotational motion. Changes in the fluorescence
lifetime of the probes used (7, 8, 14), either due to association
with other lipids or with membrane proteins, could conceivably
be responsible for the differences in fluorescence polarization
ratios between sensitive and resistant cells. In the absence of
direct measurements of fluorescence lifetime, therefore, our
results and previous results must be interpreted with caution.
However, the electron spin resonance measurements of Sieg
fried ef al. (13) also suggest that membrane lipid motion is
increased in resistant cells; these results do not suffer from this
limitation. We feel that it is safe to assert that alterations in
membrane lipid physical properties, loosely referred to as fluidity,
are associated with resistance to Adriamycin. The results pre
sented in this report also allow us to say that these alterations,
regardless of the exact nature of the motional change, are not a
direct cause of Adriamycin resistance. It is interesting, however,
that of several drug-resistant isolates tested, all exhibit an alter
ation in plasma membrane lipid structural order at early pas
sages. This may suggest that these changes have an indirect
role in drug resistance. One possible explanation is that the
development of resistance is a multistage process. Membrane
lipid changes may play a role in the early developmental stages
and would thus not be required once resistance is established.
Another possible explanation for the present findings is related
to the study of Kaufman ef al. (6), who have provided evidence
that amplified gene-containing DMS are responsible for an un
stable phase of methotrexate resistance. It thus seems possible
that DMS or some other unidentified unstable genetic element
are formed in response to cell treatment with Adriamycin. These
chromosomal elements would contain the genetic loci for the
development of both drug resistance and altered membrane
physical properties. These 2 phenotypes would be closely linked
genetically, and would appear in a high proportion, or perhaps
all, cells resistant to Adriamycin. In certain instances, such as
the R6 isolate, the DMS may be incorporated into the genome,
and thus generate a stable phenotype. In other cases such as
the R3 isolate this may not be the case, and the cells would be
unstable and undergo a reversion for membrane physical prop
erties and resistance.
4980
ACKNOWLEDGMENTS
Expert technical assistance was provided by Debra Carman and Leisa Albers.
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CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1984 American Association for Cancer Research.
VOL. 44
Involvement of Plasma Membrane Lipid Structural Order in
Adriamycin Resistance in Chinese Hamster Lung Cells
David A. Rintoul and Melvin S. Center
Cancer Res 1984;44:4978-4980.
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