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(CANCER RESEARCH 50. 1426-1430. March 1. 1990]
Mechanisms of Multidrug Resistance in HL60 Cells: Detection of Resistanceassociated Proteins with Antibodies against Synthetic Peptides That
Correspond to the Deduced Sequence of P-Glycoprotein1
David Marquardt, Sue McCrone, and Melvin S. Center2
Division of Biology, Kansas Slale University, Manhattan, Kansas 66506
ABSTRACT
HL60 cells isolated for resistance to Adriamycin are multidrug resist
ant and defective in the cellular accumulation of drug. These cells do not
however overexpress mdr\ and do not contain detectable levels of Pglycoprotein. In the present study we have prepared antisera against
synthetic peptides that correspond to various sequence domains of Pglycoprotein and have examined by Western blot analysis the reactivity
of these antisera with proteins contained in membranes of III (ill \dr
cells. All antisera are highly reactive with a M, 180,000 (plSO) Pglycoprotein contained in membranes of HL60 cells isolated for resistance
to vincristine (HL60/Vinc). In contrast, of 13 antisera tested 12 do not
react with any resistance-associated protein in the III 60 Adr isolate.
One antiserum (ASP14) is however highly reactive with a M, 190,000
protein (pl90) contained in III.60. Vdr membranes. This protein is not
detected in drug-sensitive cells. ASP14 also reacts with proteins p 195
and pSO contained in a second independent III.60/Adr isolate. Analysis
of membrane subfractions shows that pl90 is located primarily in the
endoplasmi reticulum with only low levels contained in plasma mem
branes. Additional studies demonstrate that endoplasmic reticulum of
III 6(1 Vdr cells contain a major V/, 190,000 protein that is capable of
binding the photoaffinity agent 8-azido|a-"P|ATP. p 195 contained in a
second 111.(.IIAdr isolate is also labeled with 8-azido|a-"P|ATP.
These results thus demonstrate that antiserum against a specific Pglycoprotein sequence detects a pl90 (pl95) resistance-associated mem
brane protein in two independent III 6(1 Adr isolates. pl90 (pl95) and
P-glycoprotein thus contain a minor sequence homology and based on
the specificity of ASP14 this occurs in a region which may be involved
in nucleotide binding. Possibly this sequence is common to and essential
for the functionality of proteins which contribute to resistance by reducing
cellular drug levels.
INTRODUCTION
Experimental cell lines treated with cytotoxic drugs such a
colchicine, Adriamycin, actinomycin D, or vincristine fre
quently develop resistance to the selecting agent as well as
resistance to structurally unrelated compounds (1, 2). This
multidrug resistance in many isolates occurs as a result of
reduced accumulation of drug that seems to be related to an
enhanced drug efflux system (3, 4). Reduced drug levels are
frequently related to overexpression of P-glycoprotein, a surface
membrane phosphoglycoprotein with a molecular weight of
150,000-180,000 (5-8). A family of genes termed mdrcode for
P-glycoprotein (9-12) and in human cell lines there appear to
be two members of this family mdr\ (12) and mdr3 (10, 13).
Only mdr\ has been found to be overexpressed in resistant cell
lines (13), whereas expression of mdr3 sequences has been
detected in human liver (13).
Recently HL60 cells selected for resistance to Adriamycin
have been isolated and characterized (14, 15). These cells are
multidrug resistant and defective in the cellular accumulation
Received 8/17/89; revised 11/8/89; accepted 11/27/89.
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.
1This investigation was supported by Research Grant CA-37585 from the
National Cancer Institute. Department of Health and Human Services, and by a
grant from Bristol-Myers.
2To whom requests for reprints should be addressed.
of drug (14, 15). Despite this phenotype, isolates of HL60/Adr
cells do not overexpress mdr\ ( 16) and do not contain detectable
levels of P-glycoprotein (17-19). Analysis of these cells has
revealed, however, certain membrane protein changes which
may contribute to drug resistance. Thus it has been found that
development of resistance is accompanied by a major increase
in the phosphorylation levels of a M, 150,000 membrane pro
tein which is contained in sensitive cells (18, 20). It has also
been observed that membranes of HL60/Adr cells contain a
resistance-associated M, 190,000 ATP-binding protein (16). In
the present study we have extended these findings and have
examined the possibility that proteins which contribute to drug
resistance in HL60/Adr cells may have some sequence homol
ogy with P-glycoprotein. Interest in this possibility was derived
from the finding that both p 1901 (16) and P-glycoprotein (21)
are capable of binding ATP and thus may contain similar
sequences at the nucleotide-binding site. Thus in the present
study we have prepared antisera against synthetic peptides
which correspond to the deduced sequence of human P-glyco
protein (12) and have examined the reactivity of this material
with membrane proteins of HL60/Adr cells. Of several antipeptide sera studied one has been found which reacts with a M,
190,000-195,000 protein in two independent HL60/Adr iso
lates.
MATERIALS
AND METHODS
Cells. HL60 cells were isolated for resistance to Adriamycin (HL60/
Adr) or vincristine (HL60/Vinc) as described previously (15, 17). The
HL60/Adr and HL60/Vinc isolates exhibit an 80- and 100-fold increase
in resistance to the selecting agents, respectively. A second independent
HL60/Adr isolate (14) exhibiting a 100-fold increase in resistance to
Adriamycin was generously provided by Dr. Alex Hindenburg.
Peptide Synthesis and Immunization. Peptides were synthesized ac
cording to the deduced sequence of human P-glycoprotein (12) by the
Marglin and Merrifield solid state method (22). The sequence of the
peptides and the regions of P-glycoprotein to which these sequences
correspond are given in Table 1. Synthetic peptides containing lysine
at the amino terminus were conjugated by means of glutaraldehyde to
keyhole limpet hemocyanin. The conjugated material (0.3-1.0 mg) was
emulsified with Freund's complete adjuvant and thereafter injected s.c.
into a New Zealand White rabbit. After 3 weeks a second injection of
conjugated material in incomplete adjuvant was made and after an
additional 2 weeks the rabbit was bled. Antisera were tested for reactiv
ity against synthetic peptides by using a radioimmunoassay procedure
essentially as described by Mumby et al. (23), except that the I25Ilabeled protein A was used to detect antibody binding to peptide.
Immunoblots. Membrane proteins (50 Mg)were separated by electrophoresis in a 7.5% sodium dodecyl sulfate polyacrylamide gel (24) and
the proteins were transferred to nitrocellulose paper as described by
Towbin et al. (25). The paper was incubated in PNBT for 2 h at 37°C.
The paper was thereafter incubated with antisera diluted in PNBT for
15 h at room temperature. After washing with PNBT the paper was
incubated with I25l-labeled protein A (5 x 10' cpm/ml) for 2 h at room
temperature. After extensive washing the paper was dried and iinmn
3The abbreviations used are: pl90. A/, 190,000; other proteins are similarly
designated; 8-azido[a-"P]ATP, AzAT"P; PNBT, 0.02 M phosphate buffer (pH
7.3)-O.I5 M NaCl-1% bovine serum albumin-0.05% Tween 20.
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DETECTION OF RESISTANCE-ASSOCIATED
Fig. 1. Reactivity of antipcptidc sera with
membrane proteins of sensitive and resistant
cells. Membranes were prepared from sensitive
cells (Lane I), HL60/Adr cells (Lane 2), or
HL60/Vinc cells (Lane 3), and Western blot
analysis was carried out as described in "Ma
terials and Methods," using antisera ASPI (A).
ASP9 (B), or ASP21 (C). The sequences of
the peptides to which the corresponding antisera are directed are given in Table I. Ordinate.
molecular weight in thousands.
1
180-
2
PROTEINS IN HL60/Adr CELLS
B
2
3
3
I
2
3
f
1234
Table I Reactivity of antipeptide sera with proteins of drug-resistant HL60 cells
reactivity p
Pepiide
P-glycoprotein''
190+++++++++++
sequence*GEMTD1FANAGNLEDLMSFANAGNLEDLMSN1TSDINDTGFFMNLEEDLNTRLTDDVSKINEVVSWFDDPKNTTGALTSGQALKDKKELEGAGNVTFGEVVFNYPTRP
no."129233811412117IS914612Peptide
amino
acids73-9079-9396-110171-185801-815880-8941034-10481049-10631143-11571158-11721173-11871201-12151266-1280Antisera
0
0
+++
" Peptide number corresponds to original laboratory designation.
* Peptide sequence corresponds to the deduced mdr\ P-glycoprotein sequence
(12).
f Antisera reactivity with P-glycoprotein or P190 was determined with mem
branes of HL60/Vinc or HL60/Adr cells, respectively, using Western blot analy
sis.
noreactive proteins were detected by autoradiography.
Cell Membranes. Membranes were isolated from sensitive and re
sistant HL60 cells as previously described (26). For certain experiments
plasma membranes and cndoplasmic reticulum were isolated after
centrifugation of a crude membrane fraction in a discontinuous sucrose
density gradient (26).
Labeling Membrane Proteins with AzAT'2P. Membrane proteins were
labeled with the photoaffinity agent AzAT"P (8.6 Ci/mmol, purchased
from 1CN) as described previously (16). Labeling conditions used for
each experiment are given in the figure legends. Radioactively labeled
proteins were analyzed after polyacrylamide gel electrophoresis and
autoradiography.
50-
RESULTS
Analysis of Membrane Proteins of HL60/Adr Cells Reactive
with Antisera to P-Glycoprotein Peptide Sequences. Antibodywas prepared against synthetic peptides which correspond to
the deduced sequence of P-glycoprotein (12) and the reactivity
of this material with membrane proteins of sensitive, HL60/
Adr and HL60/Vinc cells was examined by Western blot analy
sis. The HL60/Vinc isolate has previously been shown to overexpress marl (16) and to contain high levels of a M, 180,000
P-glycoprotein (17, 20). The results of a screening assay with
isolated membranes using 3 different antipeptide sera are shown
in Fig. 1. All three sera are highly reactive with the M, 180,000
P-glycoprotein contained in the HL60/Vinc isolate (Fig. 1,
Lane 3\ Table 1). Serum against peptide 1 (Table 1) also reacts
with a M, 110,000 protein contained in these cells (Fig. IA,
Lane 3). The nature of pi 10 and its involvement in drug
resistance is not known at the present time. In contrast to the
results obtained with HL60/Vinc cells the three antisera do not
react with any protein contained in membranes of sensitive cells
(Fig. 1, Lane J) or the HL60/Adr isolate (Fig. 1, Lane 2).
Further studies with 10 additional antipeptide sera show that
all are reactive with a single M, 180,000 protein contained in
Fig. 2. Reactivity of ASPI 4 with membrane proteins of sensitive and resistant
cells. Membranes were prepared from sensitive cells (Lane /), HL60/Adr cells
(Lane 2). a second HL60/Adr isolate (14) (Lane J). and HL60/Vinc cells (¡Mne
4), and Western blot analysis was carried out as described in "Materials and
Methods." using ASP 14. Ordinate, molecular weight in thousands.
membranes of HL60/Vinc cells (Table 1). In contrast, of these
10 sera 9 do not react with any resistance-associated protein in
HL60/Adr cells (Table 1). It has been found, however, that one
antipeptide serum, ASP 14 (Table 1), is highly reactive with Pglycoprotein of HL60/Vinc cells (Fig. 2, Lane 4) and with a M,
190,000 protein contained in membranes of the HL60/Adr
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DETECTION OF RESISTANCE-ASSOCIATED PROTEINS IN HL60/Adr CELLS
1234
190-
190-
Fig. 3. p 190 distribution in membrane subfractions. Plasma membranes and
endoplasmic reticulum were prepared from sensitive and HL60/Adr cells as
described in "Materials and Methods." Membrane proteins were separated by
polyacrylamide gel electrophoresis and Western blot analysis was carried out with
the ASPI 4 antiserum (Table 1). Lanes I and 3, endoplasmic reticulum of sensitive
and resistant cells, respectively. Lanes 2 and 4, plasma membranes of sensitive
and resistant cells, respectively. Ordinate, molecular weight in thousands.
isolate (Fig. 2, Lane 2). This antiserum also reacts with two
resistance-associated proteins p 195 and p50 present in mem
branes of a second independent Adriamycin-resistant isolate
(14) (Fig. 2, Lane 3). The structural relationship between pl95
and p50 is not known at the present time, pi90 (pi95), p50, or
p 180 are not detected in drug-sensitive cells with the ASP 14
serum (Fig. 2, Lane ]). The antiserum does, however, react
with some low-molecular-weight proteins contained in both
sensitive and resistant cells (Fig. 2).
Experiments have also been carried out in which plasma
membranes and endoplasmic reticulum were prepared from
HL60/Adr cells and the presence of p 190 in these membrane
fractions was examined by Western blot analysis with ASP 14.
The results demonstrate that p 190 is located primarily in the
endoplasmic reticulum (Fig. 3, Lane 3) with only barely detect
able levels in the plasma membranes (Fig. 3, Lane 4). In parallel
Fig. 4. AzAT32P labeling of membrane proteins. A crude membrane fraction
from sensitive and HL60/Adr cells was separated into plasma membranes and
endoplasmic reticulum (26), and proteins were labeled with AzAT3!P as described
in "Materials and Methods." Incubations with AzAT3IP were for 2 min, followed
by exposure of the samples to UV irradiation for 2 min. Radioactively labeled
proteins were detected after polyacrylamide gel electrophoresis and autoradiography. Lanes 1 and 3, plasma membranes of sensitive and resistant cells, respec
tively. Lanes 2 and -I. endoplasmic reticulum of sensitive and resistant cells,
respectively. Ordinate, molecular weight in thousands.
experiments with sensitive cells p 190 is not detectable in either
the endoplasmic reticulum (Fig. 3, Lane 1) or plasma mem
branes (Fig. 3, Lane 2). A. lower-molecular-weight protein with
a molecular weight of 85,000 which is contained in endoplasmic
reticulum of both sensitive and resistant cells has been found
to be reactive with ASP 14 (Fig. 3, Lanes 1 and 3).
Analysis of Membrane AzAT32P-binding Proteins. Previous
studies have shown that membranes of HL60/Adr cells contain
a M, 190,000 protein which is highly labeled with the photoaffinity agent AzAT32P (16). In view of this finding it was of
interest to determine if this protein corresponds to p 190 which
is reactive with ASP 14. To examine this, plasma membranes
and endoplasmic reticulum were isolated from sensitive and
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DETECTION OF RESISTANCE-ASSOCIATED
PROTEINS IN HL60/Adr CELLS
resistant cells and thereafter labeled with AzAT'2P as described
in "Materials and Methods." The radioactively labeled proteins
I 2 3
were analyzed after polyacrylamide gel electrophoresis. The
results demonstrate that AzAT"P labels a resistance-associated
M, 190,000 protein which is located exclusively in the endoplasmic of the HL60/Adr isolate (Fig. 4, Lane 4). With this
labeling procedure p 190 is not detected in the plasma mem
brane fraction of resistant cells (Fig. 4, Lane 3). There is
essentially no detectable labeling of p 190 with AzAT'2P in
either plasma membranes (Fig. 4, Lane 1) or endoplasmic
reticulum (Fig. 4, Lane 2) in drug-sensitive HL60 cells. The
results of this experiment and those of Fig. 3 thus demonstrate
that the membrane distribution of AzAT32P-labeled p 190 cor
responds exactly to that of pl90 reactive with ASP14. It is thus
indicated that p 190 reactive with ASP 14 is an ATP-binding
protein. AzAT32P-labeling experiments have also been carried
out with membranes of a second independent HL60/Adr isolate
(14). Membranes of these cells contain pl95 and p50 which are
reactive with ASP 14 (Fig. 2). Incubation of membranes with
AzAT32P results in the labeling of a resistance-associated pro
i
tein with a molecular weight of 195,000 (Fig. 5, Lane 1). A
protein, with molecular weight of 50,000 is also labeled to a
greater extent than a protein of similar molecular weight con
tained in drug-sensitive cells (Fig. 5, Lanes 1 and 3). The
increased labeling of p50 of resistant cells has been detected
only under conditions in which incubations with AzAT32P are
carried out for time periods of about 10-20 s. During longer
incubation periods the levels of p50 labeling in sensitive and
resistant cells are similar. Under the conditions of these exper
iments p 190 of HL60/Adr membranes is also labeled with
AzAT32P but the extent of labeling is somewhat less than that
found for p 195 (Fig. 5, Lanes 1 and 2). The results of these
studies thus suggest that the ASP14-reactive protein p 195 is
capable of binding ATP. p50 which also reacts with this antiserum may also be an ATP-binding protein. Experiments have
been carried out to immunoprecipitate AzAT12P-labeled p 190
with ASP 14. Thus far we have not detected immunoprecipitation of radioactively labeled protein. Further studies suggest
however that the ASP 14 serum does not react with native
proteins in immunoprecipitation experiments.
DISCUSSION
HL60 cells isolated for resistance to Adriamycin are defective
in the cellular accumulation of drug and this occurs as a result
of enhanced levels of an energy-dependent efflux system (14,
15). Recently a M, 190,000 resistance-associated ATP-binding
protein has been detected in membranes of these cells, suggest
ing the possibility that this protein functions in the drug efflux
pathway of this isolate (16). Several lines of evidence suggest
that p 190 is structurally distinct from P-glycoprotein. Thus,
HL60/Adr cells do not overexpress mdr\ (16, 19) and the Pglycoprotein monoclonal antibody C219 (27) does not react
with any protein in membranes of HL60/Adr cells (17, 18, 20).
In the present study we have extended these findings and have
examined in greater detail possible sequence homology of p 190
Fig. 5. AzAT3!P labeling of membrane proteins. Membranes were isolated
and P-glycoprotein. To approach this problem we have pre
from two independent HL60/Adr isolates (Lanes I and 2) and from sensitive
pared antisera against peptides with correspond to the deduced
cells (Lane 3), and proteins were labeled with AzAT32P as described in "Materials
and Methods." Results shown in Lane 1 were obtained with an isolate prepared
sequence of P-glycoprotein (12) and have examined the reactiv
by liliali;! el al. (14). Incubations with Az.AT3!P were for 10 s, followed by
ity of this material with membrane proteins in drug-resistant
exposure of the samples to UV irradiation for 2 min. Radioactively labeled
HL60 cells. Thirteen antipeptide sera have been prepared, all
proteins were detected after poKan\l.imidc gel electrophoresis and autoradiography. Exposure to AzAT"P was for 10 s, since under these conditions we can
of which are highly reactive with P-glycoprotein contained in
detect labeling of a P50 protein contained in an HL60/Adr isolate (Lane I).
HL60 cells isolated for resistance to vincristine. Screening
Ordinate, molecular weight in thousands.
assays using Western blot analysis show that 12 of these antisera are not reactive with any resistance-associated protein in
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DETECTION OF RESISTANCE-ASSOCIATED
HL60/Adr cells. One antipeptide serum, ASP14, was found
however to be highly reactive with a M, 190,000 protein con
tained in membranes of two independent HL60/Adr isolates.
The basis of the slight difference in the molecular weights of
the proteins is unknown but may be related to higher levels of
glycosylation of p 195. It was also found in these studies that
one HL60/Adr isolate contains a p50 protein which reacts with
ASP 14. The nature of this protein and its structural and func
tional relationship with pi90 (pi95) remains to be determined.
The finding that ASP 14 reacts with resistance-associated
p 190 (p 195) and P-glycoprotein suggests that these proteins
share a common sequence contained in peptide 14. At the
present time it is not known if the complete P-glycoprotein,
peptide 14 sequence is contained in p 190 (p 195). It is indicated,
however, from hybridization studies with mdrl (16) and studies
with many antipeptide sera directed against a variety of se
quence domains of P-glycoprotein (Table 1) that peptide 14
may be the only sequence shared by p 190 (p 195) and P-glyco
protein. This raises the possibility that the sequence of peptide
14 is highly conserved in proteins which contribute to resistance
by reducing cellular drug levels in an energy-requiring reaction.
This would be consistent with the evidence which suggests that
the peptide 14 sequence includes part of a nucleotide-binding
region. Thus peptide 14 has limited sequence homology with
nucleotide-binding sites detected in other proteins (28) and
proteins reactive with ASP 14 such as P-glycoprotein and p 190
(p 195) are capable of binding the photoaffmity agent AzAT"P.
P50, a protein reactive with ASP 14 and contained in a single
HL60/Adr isolate, may also be an ATP-binding protein. These
results taken together therefore suggest that ASP 14 is directed
against part of a nucleotide-binding sequence and that this
sequence may be highly conserved in proteins involved in multidrug resistance. This site may thus be of major importance to
the function of these proteins.
Previously we have shown that HL60 cells isolated for resist
ance to Adriamycin do not contain amplified mdr\ or mdri
and that these sequences are not overexpressed in the resistant
cells (16). These results along with our immunological studies
have provided strong evidence that p 190 (p 195) is encoded by
a new multidrug-resistance gene which is not genetically related
to mdr\ (12) or mdrl (10, 13). In view of this finding ASP14
may be of considerable value in analyzing multidrug-resistant
cells. Thus the serum may be important in detecting resistanceassociated proteins in experimental cell isolates which do not
contain P-glycoprotein but which exhibit reduced accumulation
of drug (29). ASP 14 may also be of value in identifying new
proteins which contribute to resistance in tumor cells from
patients undergoing chemotherapy. Finally, this antipeptide
serum should be useful as a probe in the isolation of a P190
complementary DNA.
ACKNOWLEDGMENTS
The authors wish to thank Dr. Alex Hindenburg for providing an
HL60/Adr isolate for use in these studies.
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Mechanisms of Multidrug Resistance in HL60 Cells: Detection of
Resistance-associated Proteins with Antibodies against
Synthetic Peptides That Correspond to the Deduced Sequence
of P-Glycoprotein
David Marquardt, Sue McCrone and Melvin S. Center
Cancer Res 1990;50:1426-1430.
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Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1990 American Association for Cancer Research.