[CANCER RESEARCH
45, 6088-6092,
December 1985]
Evidence for the Involvement of Two Distinct Membrane Proteins in Adriamycin
Resistance in Chinese Hamster Lung Cells1
Wallace Marsh and Melvin S. Center2
Division of Biology, Kansas State University, Manhattan, Kansas 66506
ABSTRACT
Chinese hamster lung cells resistant to Adriamycin were la
beled with inorganic [32P]orthophosphate and thereafter incu
bated with low levels of A/-ethylmaleimide. Plasma membranes
and endoplasmic reticulum were isolated and the phosphorylated
proteins were analyzed after polyacrylamide gel electrophoresis.
The results demonstrate that both plasma membranes and en
doplasmic reticulum from resistant cells contain two highly phos
phorylated proteins [M, 180,000 (p180) and M, 220,000 (p220)]
which are present in very low levels in these membrane fractions
prepared from drug sensitive cells. p220 is present in much
higher levels in the endoplasmic reticulum as compared to the
plasma membranes whereas p180 is equally distributed in these
two membrane fractions. When resistant cells revert to drug
sensitivity there is a parallel loss in the phosphorylation levels of
p180 and p220. Labeling of membrane proteins with 125Iin the
presence of chloramine-T also reveals that p180 and p220 are
present in significantly greater levels in resistant membranes as
compared to similar fractions prepared from drug sensitive cells.
Partial digests of phosphorylated p180 and p220 produced with
chymotrypsin or V8 protease reveal that each protein has a
distinct phosphopeptide pattern. Both p180 and p220 are phos
phorylated exclusively at serine residues. The results of this
study therefore suggest that resistance to Adriamycin in Chinese
hamster lung cells requires the involvement of two distinct pro
teins which are both bound to cell membranes.
Despite extensive studies p180 has been the only protein
which has thus far been identified as having an involvement in
cell resistance to Adriamycin. Recently we have found, however,
by analyzing in vitro phosphorylation of proteins in isolated
membranes that resistant cells contain three proteins with mo
lecular weights of 180,000, 220,000, and 20,000 which are not
present in membranes from cells sensitive to drug.4 All three
proteins were found to be essentially absent in membranes from
cells which have reverted to drug sensitivity. These studies thus
suggest that Adriamycin resistance may require the involvement
of multiple protein components. In the present study we have
identified p220 using in vivo labeling techniques and have ob
tained further evidence that both p220 and p180 are involved in
cell resistance to Adriamycin.
MATERIALS AND METHODS
Cells. Chinese hamster lung cells (HT-1 ) resistant to Adriamycin were
isolated as described previously (1). The cell resistant isolate R-PC4 was
cloned in soft agar before use in these studies. The revertan! cell isolate
PC4C12 was obtained as described previously (2). The resistant isolate
PC4 and the revenant are about 200- and 20-fold, respectively, more
resistant to Adriamycin than are the parent HT-1 cells. All cells were
cultured in Dulbecco's modified Eagle's medium supplemented with 10%
fetal calf serum.
Effect of NEM on Protein Phosphorylation. Sensitive, resistant, or
revertant cells were grown in 100-mm dishes in Dulbecco's modified
Eagle's medium containing 10% fetal calf serum for 48 h. The medium
was thereafter removed and 1 ml of TG medium containing 50 ^Ci of
32P;was added to each dish. After a 1-h labeling period at 37°C the
INTRODUCTION
Previous studies have shown that Chinese hamster lung cells
resistant to Adriamycin contain a plasma membrane phosphoglycoprotein (pi 803) which is not detected in cells sensitive to drug
(1-3). This protein is present in only very low levels in cells which
have reverted to drug sensitivity suggesting a close correlation
between the presence of p180 and the drug resistant phenotype
(2). It has also been observed that when resistant cells are
incubated with NEM there is a major increase in drug uptake
and a concomitant increase in the phosphorylation of p180 (2,
3). Similar changes in drug uptake and p180 phosphorylation
have also been observed in resistant cells treated with trifluoperazine or verapamil (4). Based on these findings it has been
suggested that phosphorylation of p180 plays an important role
in regulating the biological activity of this protein.
Received 2/20/85; revised 8/21/85; accepted 8/22/85.
' This investigation was supported by Research Grant CA-37585
from the
National Cancer Institute, Department of Health and Human Services.
2 To whom requests for reprints should be addressed.
3 The abbreviations used are: p180, M, 180,000 plasma membrane phosphoglycoprotein (other proteins are designated similarly); NEM, W-ethylmaleimide; TG
medium, 0.05 M Tris-HCI (pH 7.6), 0.15 M NaCI, 5 mm KCI, 0.5 rnw MgCI2, 1 mM
CaCI2, 5.5 mM D-glucose, and 1 x minimal essential medium amino acids and
vitamins.
CANCER
medium was removed and the cells were washed once with TG medium.
To each dish 3 ml of TG medium were added followed by the addition
of NEM. The cells were incubated for 6 min after which time plasma
membranes and endoplasmic reticulum were prepared as described
previously (3). The isolated membranes were characterized by analying
marker enzymes (3) and by examining thin sections in the electron
microscope. The two methods indicated that the isolated membranes
were essentially free of contaminating organelles. The phosphorylated
proteins in the isolated membrane fractions were analyzed after electro
phoresis in 7% polyacrylamide gels (5). Labeled proteins were detected
by autoradiography.
lodination of Membrane Proteins. Isolated plasma membranes or
endoplasmic reticulum were dissolved in 1% sodium dodecyl sulfate and
the proteins were labeled with 125I by the chloramine-T method as
described by Frost and Bourgaux (6).
Phosphoamino Acid Analysis. Membranes from resistant cells prelabeled with 32P,and incubated with 80 MM NEM for 6 min were isolated
and the proteins were resolved by polyacrylamide
One lane of the gel was dried and an autoradiogram
autoradiogram was used for locating p180 or p220
containing these proteins were incubated in 1.0 ml
4W. Marsh and M. Center. In vitro phosphorylation
gel electrophoresis.
was prepared. This
in the gel. Gel slices
of ammonium bicar-
and the identification of
multiple protein changes in membranes of Chinese hamster lung cells resistant to
Adriamycin, submitted for publication.
RESEARCH VOL. 45 DECEMBER
1985
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PROTEIN CHANGES
IN ADRIAMYCIN
RESISTANT
boriate (pH 8.0) containing 25 ^g of trypsin. Incubation was continued
for 15 h at 37°Cand the gel slices were removed by centrifugation. The
supernatant was evaporated to dryness and the sample was hydrolyzed
in 0.1 ml of 6 N HCI in tubes sealed under vacuum for 2 h at 105°C.The
hydrolysate was evaporated to dryness and the sample was taken up in
10 M! of a solution containing phosphoserine, phosphothreonine, and
phosphotyrosine and electrophoresed on cellulose thin layer plates in
acetic acid:pyridine:water, 50:5:945 (pH 3.5), for 2 h at 600 V. Markers
were visualized with ninhydrin spray and 32P-labeled phosphoamino acids
were visualized by autoradiography.
Phosphopeptide Analysis. Peptide mapping of phosphorylated p180
and p220 was carried out essentially as described by Cleveland et al.
(7). The gel slice containing the appropriate protein was transferred to
the top of a 15% polyacrylamide gel and thereafter incubated with 25 ^g
of chymotrypsin or 5 M9 of V8 protease for 40 min. After incubation
electrophoresis was carried out and the phosphorylated peptides were
detected by autoradiography.
RESULTS
Effect of N-Ethylmaleimide
on Protein Phosphorylation in
HAMSTER
LUNG CELLS
autoradiography. Plasma membranes from Adriamycin resistant
cells contain two labeled proteins, p180 and p220, which are
essentially absent in plasma membranes from drug sensitive
cells (Fig. 4, C and D). In the plasma membrane fraction from
resistant cells, however, p220 is found in only very low levels
(Fig. 40). Analysis of labeled proteins in the resistant endoplasmic
reticulum reveals the presence of p220 which is present in
significantly greater levels than is a similar migrating protein from
drug sensitive endoplasmic reticulum (Fig. 4, A and B). In some
instances an 125l-labeledM, 240,000 protein is present in resistant
endoplasmic reticulum in levels higher than that found in this
membrane fraction from drug sensitive cells (Fig. 4, A and B).
This finding is not, however, consistent with each experiment,
thus suggesting that the M, 240,000 protein has no relation to
the drug resistant phenotype. With the labeling technique used
p180 is also found to be contained in the endoplasmic reticulum
isolated from drug resistant cells. Under these conditions p180
is, however, a minor component and in Fig. 48 this protein is
obscured since the autoradiogram was overexposed in order to
clearly demonstrate the presence of p220. As shown in Fig. 4£
p220 and p180 labeled with 125Iare found to comigrate with 32P-
Membranes of Drug Resistant Cells. Drug sensitive, resistant,
or revertant Chinese hamster lung cells were prelabeled with 32P¡ labeled p180 and p220.
Phosphopeptide Analysis of p 180 and p220. Drug resistant
and thereafter incubated with 40 MM NEM for 6 min. Cell mem
cells were prelabeled with 32Piand thereafter incubated for 6 min
branes were prepared and the phosphoproteins were analyzed
with 80 MMNEM. A membrane fraction containing plasma mem
after polyacrylamide gel electrophoresis. Plasma membranes and
branes and endoplasmic reticulum was prepared and p180 and
endoplasmic reticulum prepared from drug resistant cells contain
p220 were resolved by polyacrylamide gel electrophoresis. The
two highly phosphorylated proteins, p180 and p220, which are
isolated proteins were partially digested with chymotrypsin or V8
present in greatly reduced levels in membranes from sensitive
protease and analyzed according to the method of Cleveland ef
cells (Fig. 1). Membranes from cells which have reverted to drug
al. (7). An analysis of p180 and p220 digested with either
sensitivity also have greatly reduced levels of phosphorylated
p180 and p220 (Fig. 1, C and F). The levels of these proteins in chymotrypsin or V8 protease shows that the two proteins have
distinct phosphopeptide patterns. Digestion of p180 with chy
revertant cells are essentially the same as those found in the
motrypsin results in at least four closely migrating phosphopep
parent Adriamycin sensitive cell. Experiments of this type have
tide components (Fig. 5A). p220, in contrast, digested under
been repeated several times and p220 and p180 have been
these
same conditions contains only a single major phosphory
found to be the only two phosphoproteins which appear in high
levels in resistant membranes but at greatly reduced levels in lated peptide (Fig. 46). Digests of p180 treated with V8 protease
contains four major and three minor phosphopeptides (Fig. 5C)
membranes from sensitive or revertant cells. At higher levels of
while p220 contains only a single major phosphopeptide com
NEM (80 MM)a similar phosphoprotein pattern in resistant memponent (Fig. 5D).
banes is observed and the levels of these proteins in drug
Phosphoamino Acid Analysis. A membrane fraction contain
sensitive cells are still extremely low (Fig. 2). Identical results
have also been obtained when 32Prprelabeled cells are treated
ing plasma membranes and endoplasmic reticulum was prepared
from 32P(prelabeled resistant cells treated with 80 UM NEM for 6
with 160 and 320 MMNEM for 6 min. The results of these studies
min. p180 and p220 were resolved by polyacrylamide gel elec
also demonstrate that p220 is located primarily in the endo
trophoresis and the phosphoamino acids of the proteins were
plasmic reticulum fraction whereas p180 is distributed about
determined as described in "Materials and Methods." The results
equally in the plasma membranes and endoplasmic reticulum.
Experiments have also been carried out in which 32Prlabeled show that both p180 and p220 are phosphorylated exclusively
at serine residues (Fig. 6). We have also observed that p180
resistant cells were incubated in the absence or presence of 80
prepared from 32Piprelabeled cells not treated with NEM is also
MM NEM and the phosphoproteins present in isolated endo
phosphorylated only at serine residues (not shown). Previously
plasmic reticulum were analyzed after polyacrylamide gel elec
we have shown that p180 prepared from 32P¡
prelabeled cells
trophoresis. Incubation of cells in the absence of NEM results in
treated with 5 mw NEM for 20 min is phosphorylated at both
the phosphorylation of p180 whereas under these same labeling
serine and threonine (2). The basis of the threonine phosphoryla
conditions p220 cannot be detected (Fig. 3A). When the cells are
prelabeled with 32Piand thereafter incubated with 80 UM NEM
tion at high NEM concentrations is not known.
for 6 min, there is an enhancement of p180 phosphorylation and
a major phosphorylation of p220 (Fig. 36).
Analysis of 125l-Labeled Membrane Proteins. Plasma mem
branes and endoplasmic reticulum were prepared from drug
sensitive and resistant cells and the proteins were labeled with
125Iin the presence of chloramine-T. The proteins were electro
phoresed in a polyacrylamide
gel and thereafter identified by
CANCER
RESEARCH
DISCUSSION
Previously we have
Adriamycin in Chinese
ence of a M, 180,000
(1-4). In the present
VOL. 45 DECEMBER
provided evidence that cell resistance to
hamster lung cells is related to the pres
plasma membrane phosphoglycoprotein
study we have identified an additional
1985
6089
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PROTEIN CHANGES
protein, p220, which
exhibit an Adriamycin
this protein is based
phosphorylated when
IN ADRIAMYCIN
RESISTANT
HAMSTER
LUNG CELLS
present in colchicine resistant cells but absent in cells sensitive
to drug (16). This protein was identified by analyzing protein
phosphorylation in isolated membranes frorrr drug resistant cells
(16). Recently monoclonal antibody has been prepared against
a specific M, 230,000 protein which is present in plasma mem
branes of cells resistant to aclacinomycin (17). Of interest is the
finding that the aclacinomycin resistant cells are also cross-
may also be required in order for cells to
resistant phenotype. The identification of
on the finding that p220 becomes highly
resistant cells are prelabeled with 32Piand
thereafter incubated with low levels of NEM for short time
periods. p220 is present in greatly reduced levels in drug sensi
tive cells and cells which have undergone a reversion to drug
resistant to Adriamycin (16). It is also of interest that a M, 19,000
sensitivity. This latter finding thus suggests a correlation between
protein has been identified which may be involved in cell resist
the presence of p220 and drug resistance.
Previously we have shown that p180 can be labeled with 32P¡ ance to vincristine (18). We have also found that p20 is present
in membranes of adriamycin resistant cells but is essentially
and that there is an enhancement in the phosphorylation of this
absent in cells sensitive to drug." This protein has been identified
protein when labeled cells are incubated in the presence of 5by analyzing proteins phosphorylated in vitro in isolated mem
10 (TIMNEM for 25 min (2, 3). Under these labeling conditions
branes. All of these results taken together suggest that the mutip220 is not detected. The reason for the absence of detectable
drug resistant phenotype exhibited by a number of different
phosphorylated p220 in our previous studies is not known.
isolates (8,10,12) may require the involvement of multiple protein
Possibly in the presence of 5 mw NEM p220 is phosphorylated
components. In cells isolated for Adriamycin resistance p180,
and rapidly dephosphorylated or alternatively the phosphorylated
p220, and perhaps p20 may fulfill this requirement.
protein may become dissociated from its membrane location.
Our studies thus suggest that in order to detect a phosphorylated
form of p220 it is essential that prelabeled cells be incubated in
low levels of NEM for short time periods. Our studies carried out
thus far indicate that in the resistant cell p180 is phosphorylated
whereas p220 is not. When the resistant cell is incubated with
NEM both proteins become highly phosphorylated. The mecha
nism by which NEM induces the selective phosphorylation of
these two proteins is not known. Recently we have found that
NEM induces drug uptake in resistant cells at 40 pM with
maximum cellular drug accumulation occurring at 80 UM. Thus
there seems to be a correlation between the concentration of
NEM required for enhancing drug uptake and the concentration
required to induce the phosphorylation of p180 and p220. These
results may suggest that the biological activity of both proteins
is regulated by phosphorylation. Thus as p180 and p220 are
phosphorylated in the presence of NEM they become biologically
inactive and Adriamycin can accumulate in the cell.
At the present time the exact structural relationship that exists
between p180 and p220 is not known. Certain lines of evidence
indicate that the two proteins are probably distinct. This is based
on the finding that the two proteins yield distinct phophopeptide
patterns after digestion with either chymotrypsin or V8 protease.
Further evidence that the proteins are distinct is suggested by
the finding that p220 is located primarily in the endoplasmic
reticulum whereas p180 is found in high levels in both the plasma
membrane and endoplasmic reticulum. Previous studies have
also shown that p180 can be radioactively labeled after incuba
tion of resistant cells with [14C]glucosamine (1). In contrast p220
REFERENCES
1. Garman, D., and Center, M. S. Alterations in cell surface membranes in Chinese
hamster lung cells resistant to Adriamycin. Biochem. Biophys. Res. Commun.,
705:157-163, 1982.
2. Center, M. S. Evidence that Adriamycin resistance to Chinese hamster lung
cells is regulated by phosphorylation of a plasma membrane glycoprotein.
Biochem. Biophys. Res. Commun., 775.-159-166, 1983.
has not been found to be labeled under these conditions.
Proteins having a molecular weight similar to that of p180
have been identified in cell lines isolated for resistance to colchicine (8), actinomycin D (9), vinblastine (10), vincristine (11), and
daunomycin (12). These M, 150,000-170,000 proteins are pres
ent in only very low levels in drug sensitive cells and cells which
have reverted to drug sensitivity (8-12). Many of these cells lines
are also cross-resistant to Adriamycin (12-15). Proteins similar
in size to p220 have also been identified in certain drug resistant
cell lines. Thus a M, 200,000 protein has been found to be
CANCER
RESEARCH
3. Garman, D., Albers, L., and Center, M. S. Identification and characterization
of a plasma membrane phosphoprotein which is present in Chinese hamster
lung cells resistant to Adriamycin. Biochem. Pharmacol., 32:3633-3637,1983.
4. Center, M. S. Mechanisms regulating cell resistance to Adriamycin: evidence
that drug accumulation in resistant cells is modulated by phosphorylation of a
plasma membrane glycoprotein. Biochem. Pharmacol., 34: 1471-1476,1985.
5. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head
of bacteriophage T4. Nature (Lond.), 227: 680-685, 1970.
6. Frost, E., and Bourgaux, P. Decapsulation of polyoma virus: identification of
subviral species. Virology, 68: 245-255, 1975.
7. Cleveland, D. W., Fischer, S. G., Kirschner, M. W., and Laemmli, U. K. Peptide
mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel
electrophoresis. J. Biol. Chem., 252:1102-1106,1977.
8. Juliano, R. L., and Ling, V. A surface glycoprotein modulating drug permeability
in Chinese hamster ovary cell mutants. Biochim. Biophys. Acta, 455: 152162, 1976.
9. Peterson, R. H. F., and Siedler, J. L. Plasma membrane proteins and glycoproteins from Chinese hamster cells sensitive and resistant to actinomycin D. J.
Supramol. Struct., 9: 289-298, 1978.
10. Beck, W. T., Mueller, T. J., and Tanzer, L. R. Altered surface membrane
glycoproteins in Vinca alkaloid resistant human leukemic lymphoblasts. Cancer
Res., 39: 2070-2076, 1979.
11. Peteson, R. H. F., Meyers, M. B., Spengler, B. A., and Siedler, J. L. Alterations
of plasma membrane glycopeptides and gangliosides of Chinese hamster cells
accompanying resistance to daunorubicin and vincristine. Cancer Res., 43:
222-228,1983.
12. Kartner, N., Shales, M., Riordan, J. R., and Ling, V. Daunorubicin-resistant
Chinese hamster ovary cells expressing multidrug resistance and a cell-surface
P-glycoprotein. Cancer Res., 43:4413-4419,1983.
13. Ling, V. Drug resistance and membrane alteration in mutants of mammalian
cells. Can. J. Genet. Cytol., 77: 503-515,1975.
14. Riehm, H., and Biedler, J. L. Cellular resistance to daunomycin in Chinese
hamster cells in vitro. Cancer Res., 31: 409-412,1971.
15. Skovsgaard, T. Mechanism of cross-resistance between vincristine and dau
norubicin in Ehrlich ascites tumor cells. Cancer Res., 38: 4722-4727,1978.
16. Carlsen, S. A., Till, J. E., and Ling, V. Modulation of drug permeability in
Chinese hamster ovary cells: possible role for phosphorylation of surface
glycoproteins. Biochim. Biophys. Acta, 467: 238-250, 1977.
17. Sugimoto, Y., Suzuki, H., and Tanaka, N. Alteration of plasma membrane of
drug-resistant tumor cells: 230-kilodalton protein identified by monoclonal
antibody. Biochem. Biophys. Res. Commun., 774: 969-975,1983.
18. Meyers, M. B., and Biedler, J. L. Increased synthesis of a low molecular weight
protein in vincristine-resistant cells. Biochem. Biophys. Res. Commun., 99:
228-235, 1981.
VOL.
45
DECEMBER
1985
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D E F
'
A
B
C
D
Fig. 1. Effect of NEM on protein phosphorylation in membranes of drug sensitive, resistant, and revenant cells. Cells were prelabeled with Å“p, and thereafter incubated
with 40 put NEM for 6 min at 37°Cas described in "Materials and Methods." Plasma membranes and endoplasmic reticulum were prepared and the phosphoproteins
contained in the sensitive (Lanes A and D), resistant (Lanes B and E). and revertant (Lanes C and F) membrane preparations were analyzed after polyacrylamide gel
electrophoresis. Proteins were detected by autoradiography. Lanes A-C, endoplasmic reticulum; Canes D-F, plasma membranes.
Fig. 2. Protein phosphorylation in isolated membranes from sensitive and resistant cells. Sensitive and resistant cells were prelabeled with 32P,and thereafter incubated
with 80 JIM NEM for 6 min at 37°Cas described in "Materials and Methods." Plasma membranes and endoplasmic reticulum were prepared and the phosphoproteins in
sensitive (Lanes A and C) and resistant (Lanes B and 0) preparations were analyzed after polyacrylamide gel electrophoresis. Lanes A and B, plasma membranes; Lanes
C and D, endoplasmic reticulum.
Fig. 3. Protein phosphorylation in resistant cells incubated in the absence or presence of NEM. Drug resistant cells were prelabeled with XP( and thereafter incubated
in either the absence or the presence of 80 ¿IM
NEM for 6 min at 37°C.Endoplasmic reticulum was prepared and the phosphoproteins were analyzed after polyacrylamide
gel electrophoresis. Lanes A and B, phosphoproteins
present in membranes from cells incubated in the absence or presence, respectively, of NEM.
6091
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31, 2017. © 1985 American Association for Cancer Research.
A
A B G D
B
PSERPTHRPTYR-
Rg. 4. Analysis of membrane proteins labeled with 125I.Plasma membranes and endoplasmi reticulum were isolated from drug sensitive and resistant cells and the
proteins were labeled with 125Iin the presence of chloramine-T as described in "Materials and Methods." The labeled proteins were electrophoresed in a 7% polyacrylamide
gel and identified after autoradiography. Lanes A and B, sensitive and resistant endoplasmic reticulum, respectively; Lanes C and 0, sensitive and resistant plasma
membranes, respectively. Lane E, results of a parallel experiment in which resistant cells were prelabeled with Å“P,and treated with 80 UM NEM for 6 min. Cell membranes
were prepared and the endoplasmic reticulum fraction was electrophoresed as described above.
Fig. 5. Phosphopeptides of p180 and p220 digested with chymotrypsin or V8 protease. Resistant cells were prelabeled with 32P¡
and thereafter incubated with 80 MM
NEM for 6 min. A membrane fraction containing both plasma membranes and endoplasmic reticulum was prepared and p180 and p220 were resolved by polyacrylamide
gel electrophoresis. The gel slices containing these proteins were treated as described by Cleveland et al. (7). Partial digestions were carried out with either chymotrypsin
(25 ng/well) (Lanes A and B) or V8 protease (5 ^g/well) (Lanes C and D). Incubations were for 40 min at room temperature. After the electrophoretic run the
phosphopeptides were detected by autoradiography. Lanes A and C, p180 digest; Lanes B and D, p220 digest.
Fig. 6. Phosphoamino acid analysis of p220 and p180. Phosphoamino acid analysis of MP, labeled p180 and p220 was determined as described in "Materials and
Methods." Lane A, p180; Lane B, p220; P-SER. phosphoserine; P-THR, phosphothreonine; P-TYR, phosphotyrosine.
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1985 American Association for Cancer Research.
Evidence for the Involvement of Two Distinct Membrane
Proteins in Adriamycin Resistance in Chinese Hamster Lung
Cells
Wallace Marsh and Melvin S. Center
Cancer Res 1985;45:6088-6092.
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