Download Drug Transport Mechanisms in HL60 Cells

[CANCER RESEARCH 52. 3157-3163, June 1. 1992|
Drug Transport Mechanisms in HL60 Cells Isolated for Resistance to Adriamycin:
Evidence for Nuclear Drug Accumulation and Redistribution in Resistant Cells1
David Marquardt and Melvin S. Center2
Division of Biology, Kansas State University, Manhattan, Kansas 66506-4901
ABSTRACT
HL60 cells isolated for resistance to Adriamycin are multidrug resist
ant and defective in the cellular accumulation of drug. These cells do not
contain detectable levels of P-glycoprotein. At the present time the
mechanism by which HL60/Adr cells reduce drug levels is not known.
To gain insight into the molecular basis of this system we have analyzed
transport pathways and the distribution of daunomycin in drug-resistant
HI/60 cells. Using a cell fractionation technique we find that the major
portion of daunomycin accumulates in the nucleus of both sensitive and
resistant cells. Further studies reveal, however, that under efflux condi
tions drug is retained in the nuclei of sensitive cells but rapidly removed
from the nuclei of the resistant isolate. Essentially identical results are
obtained when daunomycin distribution and transport are analyzed by
fluorescence microscopy. A number of agents which alter transport
processes have been tested for their effect on drug accumulation in
resistant cells. Thus we find that brefeldin A, which disassembles Golgi,
and various lysosomotropic agents such as chloroquine and methylamine
do not affect drug levels. In contrast the protonophores nigericin and
monensin induce an increase in drug accumulation and inhibit efflux.
The results of this study thus suggest that resistance in IIL60/Adr
cells is related to a mechanism whereby drug is transported to the nucleus
and thereafter rapidly redistributed to the extracellular space. The mo
lecular basis of this transport pathway is not known.
INTRODUCTION
Multidrug resistance in many experimental isolates is related
to the presence of a surface membrane P-glycoprotein (1-4).
Cell lines containing this protein are defective in the cellular
accumulation of drug, and this appears to be related to the
presence of enhanced levels of an energy-dependent drug efflux
system (5, 6). Current evidence indicates that P-glycoprotein
functions in this efflux pathway as a transporter which binds
drug (7, 8) and exports this material to the exterior of the cell.
Hydrolysis of ATP by the P-glycoprotein ATPase activity (9)
may provide the energy which drives this reaction.
Recently HL60 cells selected for anthracycline resistance
have been isolated and characterized (10, 11). These cells ex
hibit a multidrug-resistant phenotype, are defective in the cell
ular accumulation of drug, and contain enhanced levels of a
drug efflux system (10-12). Despite these properties isolates of
these cells do not overexpress mdrl (13, 14) and do not contain
detectable levels of P-glycoprotein (12, 15). Proteins contained
in cell membranes of these isolates which may contribute to
drug resistance have been described, however. Thus it has been
observed that with cell surface labeling techniques a M,
150,000-160,000 protein (PI50) can be detected in resistant
but not sensitive cells (10, 11). Other studies show that devel
opment of resistance is accompanied by a major increase in the
Received 9/9/91; accepted 3/25/92.
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.
' Supported by Research Grant CA37585 from the National Cancer Institute.
Department of Health and Human Services, and by a grant from Bristol-Myers
Squibb.
2 To whom requests for reprints should be addressed, at Division of Biology,
Ackert Hall. Kansas State University. Manhattan, KS 66506-4901.
phosphorylation levels of P150 and that this protein may ac
tually represent a hyperphosphorylated form of a protein con
tained in sensitive cells ( 12). It has also been found that resistant
isolates contain increased levels of a M, 190,000 membrane
protein (PI90) which is capable of binding ATP (13). Recent
studies have shown that antiserum against a synthetic peptide
which corresponds to a specific P-glycoprotein sequence reacts
with both P-glycoprotein and P190 (16). This peptide contains
sequences which may be part of a nucleotide binding site in Pglycoprotein and P190 (16).
The mechanism by which HL60/Adr cells reduce cellular
drug accumulation is not known. Studies carried out thus far
have not identified a drug-binding protein which may act as a
drug transporter in the efflux pathway (13). It thus seems to be
indicated that HL60/Adr cells reduce drug levels by a mecha
nism distinct from that of isolates containing P-glycoprotein.
In the present study we have examined drug transport pathways
in HL60 cells isolated for resistance to Adriamycin.
MATERIALS
AND METHODS
Materials. [3H]Daunomycin (1.6 Ci/mmol) was purchased from New
England Nuclear. Brefeldin A was from Epicentre Technologies (Mad
ison, WI). All other agents were from Sigma.
Isolation of Resistant Cells. HL60 cells isolated for resistance to
Adriamycin (HL60/Adr) were prepared as described previously (11).
The isolate used in these studies is multidrug resistant and exhibits an
80-fold increase in resistance to the selecting agent (12).
Fractionation of Cells Which Have Accumulated pHjDaunomycin.
Cells growing in RPMI-10% fetal bovine serum were centrifuged and
thereafter suspended in fresh media at a concentration of 1 x 106/ml.
The cells were incubated with [3H]daunomycin (600 cpm/ng) for var
ious time periods at 37°Cin a COi incubator and thereafter centrifuged
and suspended in cold 0.01 M Tris-HCI (pH 7.6)-1.0 mivi MgCl2. The
cells were homogenized with 15 strokes of a glass homogenizer and
centrifuged at 500 x g for 5 min. The pellet containing the nuclei was
collected, and the supernatant was centrifuged at 10,420 x g for 45
min to pellet membranes. Total radioactivity associated with nuclei,
cytoplasm, and membranes was thereafter determined.
Fluorescence Microscopy. Sensitive and resistant cells were centri
fuged and suspended in fresh RPMI-10% fetal bovine serum at a
concentration of 1 x 106/ml. The cells were incubated with daunomycin
(0.5 /jg/ml) for 40 min at 37°Cand thereafter centrifuged and placed
in fresh drug-free media. After incubation at 37°Cfor various time
periods aliquots were taken and cytospun onto a glass slide. The cells
were examined by either phase-contrast or fluorescence microscopy
using a Nikon UFX-IIA microscope. For certain experiments live cells
in drug-free media were placed directly on a microscope slide and
examined by phase contrast or fluorescence microscopy.
Analysis of Drug Efflux in Cells Loaded with Drug in the Presence of
Azide. Cells growing in RPMI-10% fetal bovine serum were centrifuged
and suspended in efflux media containing 50 mM Tris-HCI (pH 7.6),
0.14 M NaCl, 5.0 mM KCI, 1.0 mM CaCl2, 0.5 mM MgCl2, 2 mM
glutamine, 1 x minimum essential medium amino acids and vitamins,
and 2% fetal bovine serum. Sodium azide was added to 10 mM, and the
cells were incubated with daunomycin (1 Mg/ml) for 30 min at 37°C.
At the end of this time period the cells were centrifuged and suspended
in EF media containing 15 HIM glucose. Aliquots were taken and
examined microscopically as described above.
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DRUG RESISTANCE IN HL60 CELLS
Effect of Various Agents on Drug Efflux. Cells were loaded with [3H]
daunomycin in EF media in the absence of glucose and presence of
azide as described above. At the end of this time period the cells were
centrifuged and suspended in EF media containing 15 HIMglucose. A
single agent was added, and after incubation for various time periods
the cells were centrifuged and radioactivity contained in the pellet was
determined.
Analysis of |3H]Daunomycin Metabolism. Cells in complete RPMI
were incubated with [3H]daunomycin at 37°Cfor periods of up to 2 h,
and the cell pellet was thereafter extracted with chloroform:methanol
(2:1) for 90 min on ice. Under these conditions [3H]daunomycin is
completely recovered from the cell. The chloroform:methanol extract
was evaporated to dryness, and the dried material was dissolved in 0.01
M Tris-HCl (pH 7.6). This sample was thereafter chromatographed on
silica gel thin-layer sheets in a buffer containing 65% chloroform-30%
methanol-5% water. After drying the sheet was cut into 0.5-cm strips,
and the radioactivity in each was determined and compared with a [3H]
daunomycin marker.
RESULTS
Accumulation and Distribution of |'H|Daunomycin in Sensitive
and Resistant Cells. Sensitive and HL60/Adr cells in complete
RRMI were incubated with [3H]daunomycin, and fractions
containing nuclei, cytoplasm, and membranes were prepared as
described in "Materials and Methods." Analysis of distribution
of [3H]daunomycin demonstrates that about 80% of the drug
is contained in the nuclei of both sensitive and resistant cells
(Fig. 1). The two cell types also contain similar levels of drug
in the cytoplasmic and membrane fractions (Fig. 1). Analysis
of drug accumulation under identical conditions demonstrates
that resistant cells accumulate about one-half the amount of
[3H]daunomycin as sensitive cells (not shown). In view of these
findings studies were carried out to examine rates of [3H]
daunomycin efflux from the nuclear, membrane, and cyto
plasmic fractions. In these experiments cells were loaded with
[3H]daunomycin in complete RPMI and thereafter suspended
in drug-free media. At various time periods the amount of drug
associated with various cell fractions was determined (Fig. 2).
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Fig. 1. Intracellular distribution of l'I I|ilaiinonni-in. Sensitive and resistant
HL60 cells growing in RPMI-10% fetal bovine serum were centrifuged and
suspended in fresh media. The cells (5 x 10s ml) were thereafter incubated for 45
min at 37'C with [3H]daunomycin (600 cpm/ng; final concentration, 0.4 jig/ml).
At the end of the incubation period cell fractionation was carried out, and the
distribution of radioactively labeled drug was determined as described in "Mate
rials and Methods." The amount of drug contained in nuclei, cytoplasm, and
membranes is presented as the percentage of total (3H]daunomycin accumulated.
In three identical experiments the difference in drug distribution has varied by no
more than 10%. •¿,
sensitive cells; D, resistant cells.
0
JO
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Time (min)
Fig. 2. Drug efflux from cell fractions. Sensitive (A) and resistant (B) cells
were centrifuged, suspended in fresh RPMI-10% fetal bovine serum, and there
after incubated with [3H]daunomycin for 40 min. At the end of this incubation
period the cells were centrifuged and suspended in complete RPMI, and an
aliquot of cells was fractionated into nuclei, membranes, and cytoplasm as
described in "Materials and Methods." The remaining cells were allowed to
continue incubation, and after 10, 30, and 60 min cell fractionation was carried
out. The amount of [3H]daunomycin contained in the various cell fractions during
the efflux is compared to that contained in the same fractions prepared prior to
the efflux period. O, nuclei; •¿
cytoplasm; D, membranes.
The results demonstrate a rapid and major loss of drug from
all fractions of the resistant cell (Fig. IB). Cytoplasmic levels
of drug remain relatively constant during a 30- and 60-min
efflux period. In resistant cells this may represent drug which
accumulates as it passes from the nucleus to the cytoplasmic
space. Of particular interest is the finding that drug which is
located in the nucleus of resistant cells (Fig. 2B) is rapidly
removed, whereas in sensitive cells there is essentially a com
plete nuclear retention of drug (Fig. 2A). Loss of drug does
occur, however, from the cytoplasmic and membrane fractions
of sensitive cells, but this is less than that occurring in these
same fractions from resistant cells (Fig. 2).
Analysis of Drug Distribution and Efflux Using Fluorescence
Microscopy. To gain additional evidence for a nuclear drug
accumulation in the HL60/Adr isolate, detailed studies were
carried out to analyze by fluorescence microscopy daunomycin
distribution in sensitive and resistant cells. In these experiments
cells were incubated with daunomycin, centrifuged, and there
after suspended in drug-free media. After incubation for various
time periods the cells were examined microscopically. The
results demonstrate that after a 40-min incubation period with
daunomycin major levels of drug are contained in the nuclei of
both sensitive (Fig. 3/1) and resistant cells (Fig. 3B). Incubation
of resistant cells in drug-free media for 40 min results in a loss
of daunomycin from the nuclear compartment (Fig. 3D),
whereas under these same conditions drug is retained in the
nuclei of sensitive cells (Fig. 3C). Phase-contrast microscopic
examination of cells during these experiments does not indicate
any detectable differences in sensitive and resistant cells, nor is
there any preferential loss of resistant cells during the efflux
period (Fig. 3, E and F). Additional experiments have been
carried out in which resistant cells were loaded with daunomy
cin in glucose-free media containing sodium azide. The cells
were centrifuged suspended in media containing glucose and
after incubation for various time periods aliquots were exam
ined by fluorescence microscopy (Fig. 4). Control cells exam
ined immediately after being placed in glucose media contain a
high nuclear localization of drug (Fig. 4A). Continued incuba
tion of cells for time periods of 20, 40, and 60 min results in a
progressive loss of daunomycin from the nuclei of resistant cells
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DRUG RESISTANCE IN HL60 CELLS
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Fig. 3. Analysis of drug transport using fluorescence microscopy. Sensitive (A, C, and E) and resistant (A, O, and F) cells growing in RPMI-10% fetal bovine serum
were incubated with daunomycin (0.5 jig/ml) for 40 min at 37°C.The cells were centrifuged and resuspended in complete RPMI, and a sample was cytospun onto a
glass slide and taken for microscopy (A and B) as described in "Materials and Methods." The remaining cells were incubated for 40 min (C and D), after which the
cells were examined by fluorescence microscopy. All samples were examined by phase contrast, and the results of the 40-min time point for sensitive and resistant
cells are given in fand F, respectively. In separate experiments Hoechst staining of cytospun cells was used to verify that fluorescent structures identified in these
experiments actually represent intact nuclei.
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DRUG RESISTANCE IN HL60 CELLS
(Fig. 4, B-D). In parallel experiments with sensitive cells there
was essentially complete retention of drug in the nucleus during
the 60-min efflux period (not shown). Studies have also been
carried out in which sensitive and resistant cells were loaded
with drug in complete RPMI and thereafter incubated under
efflux conditions. Cells from the flask were placed directly on
a microscope slide and examined by fluorescence microscopy
(Fig. 5). Prior to incubation under efflux conditions fluores
cence is distributed throughout the entire cell (Fig. 5, A and B).
Based on the cell fractionation studies (Fig. 1) it would be
predicted, however, that most of the drug is located in the nuclei
of sensitive and resistant cells. Analysis of the live cells under
efflux conditions demonstrates a progressive loss of drug from
the resistant isolate (Fig. 5, B, D, and F). In contrast only low
levels of drug are removed from sensitive cells (Fig. 5, A, C,
and E). These studies also demonstrate that as drug is removed
from HL60/Adr cells there is a localization and accumulation
of daunomycin at a perinuclear site (Fig. 5F). At the present
time the identity of the structure which contains daunomycin
is not known.
Extensive studies have been carried out to analyze the metab
olism of intracellular daunomycin. Using the thin-layer Chro
matographie technique described in "Materials and Methods"
we have not detected any change in the drug after incubation
with sensitive or resistant cells. These results strongly suggest
that the loss of daunomycin fluorescence from the nuclei of
resistant cells is not due to any major structural change in the
drug.
Effect of Various Agents on Drug Accumulation in Resistant
Cells. The results described above suggest that drug which
reaches the nucleus of resistant cells is rapidly removed and
redistributed to the extracellular space. To examine the possible
mechanisms involved in this process we have analyzed the effect
of a variety of agents on drug accumulation in resistant cells.
Thus we have found that the incubation of resistant cells in the
presence of brefeldin A (18 ^M) for periods of up to 2 h has no
effect on cellular drug levels (not shown). This agent has pre
viously been shown to be highly active in blocking protein
transport by a mechanism which involves the disassembly of
the Golgi apparatus (17, 18). Similarly, drug levels are not
altered by the incubation of resistant cells for periods of up to
2 h with chloroquine (90 ^M), methylamine (10 mivi), or am
monium chloride (20 HIM) (not shown). These agents are ca
pable of alkalinizing and disrupting the function of a variety of
intracellular membrane vesicles (19). In contrast to these agents
the protonophores nigericin and monensin (19, 20) induce an
increase in drug accumulation (Fig. 6A) and inhibit efflux (Fig.
6B). Nigericin at a final concentration of 6 pg/m\ induces a
complete block of [3H]daunomycin efflux from HL60/Adr cells
(Fig. 6B). We have consistently found that nigericin is consid
erably more active than monensin in inducing drug accumula
tion and inhibiting efflux. At the concentration used neither
nigericin or monensin affects drug accumulation in sensitive
cells (not shown). Studies have also been carried out to examine
the effect of nigericin on drug efflux from the nucleus. Using
fluorescent microscopic techniques we find that in the presence
Fig. 4. Analysis of drug efflux using fluorescence microscopy. Resistant cells
were loaded with daunomycin (I ng/ml) in the presence of azide as described in
"Materials and Methods." The cells were centrifuged and suspended in EF media
containing 15 HIMglucose, and a sample was taken for microscopy I (). Additional
aliquots were taken for microscopy after incubation of the cells at 37'C for 20
(B), 40 (C). and 60 (D) min.
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DRUG RESISTANCE IN HL60 CELLS
Fig. 5. Direct analysis of drug transport using fluorescence microscopy. Sensitive (A, C, and E) and resistant (fi, D, and f) cells growing in RPMl-10% fetal bovine
serum were incubated with daunomycin (0.5 ng/ml) for 40 min. The cells were centrifuged and resuspended in complete RPMI, and a sample was placed directly on
a microscope slide and examined by fluorescence microscopy (A and fi). The remaining cells were incubated for 40 (C and D) and 60 (£and F) min. and samples
were examined directly by fluorescence microscopy. Although fluorescence is distributed throughout sensitive and resistant cells during the initial 40-min incubation
with daunomycin (A and fi), fractionation studies (Fig. 1) indicate that drug is located primarily in the nucleus.
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DRUG RESISTANCE IN HL60 CELLS
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30
60
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Time (min)
150
20 4o so ao
Time (min)
Fig. 6. Effect of nigericin and monensin on drug accumulation and efflux.
Resistant cells (5 X I0'/ml) in complete RPMI were incubated for 5 min in the
absence or presence of either nigericin (6 /Jg/ml) or monensin (6 ng/ml), after
which |'H|daunomycin was added to the cells. Incubations were continued at
37°C,and at various time periods aliquots were taken and, after centifugation,
radioactivity contained in the cell pellet (A) was determined. A separate experi
ment was carried out to examine the effect of nigericin and monensin on drug
efflux (B). In this study resistant cells were loaded with [3H|daunomycin in the
presence of azide as described in "Materials and Methods." The cells were
ccntrifuged, suspended in EF media containing IS HIMglucose, and thereafter
incubated in the absence or presence of either nigericin or monensin at a final
concentration of 6 >ig/ml. At the various times indicated the radioactivity asso
ciated with the cell pellet was determined. D, control cells incubated in the absence
of agent: O, cells incubated with nigericin: •¿
cells incubated with monensin.
of nigericin there is essentially a complete retention of drug in
the nuclei of resistant cells (not shown).
DISCUSSION
Previous studies have shown that HL60 cells isolated for
resistance to anthracyclines are defective in the cellular accu
mulation of drug (10, 11). Results obtained thus far suggest
that reduced drug levels are related to an enhanced efflux system
(10, 11). In the present study we have extended our previous
findings and have analyzed intracellular pathways for drug
transport in the resistant cell. Using both cell fractionation and
fluorescence microscopy we have shown that incubation with
daunomycin results in a major accumulation of drug in the
nuclei of both sensitive and resistant cells. The nuclei of sensi
tive cells retain the drug, where it would be expected to exert a
cytotoxic effect. In contrast resistant cells contain a mechanism
whereby drug which reaches the nucleus is redistributed to the
extracellular space. Extensive studies suggest that this mecha
nism does not involve any structural change in the drug. At the
present time the exact pathway taken by the drug as it moves
from the nucleus to the exterior of the cell is not known. Direct
examination of live resistant cells by fluorescence microscopy
indicates that under efflux conditions drug localizes at a site
which appears to be in close proximity to the nucleus. The
nature of this site and its involvement in efflux is not known at
the present time, however. Hindenburg et al. (21) have previ
ously analyzed daunomycin distribution in an independent iso
late of HL60/AR cells. Examination of these cells using digi
tized video fluorescence microscopy and confocal microscopy
indicates that daunomycin localizes at a perinuclear site which
possibly represents a prelysosomal sorting compartment (21,
22). Similarly, Willingham et al. (23) have utilized fluorescence
microscopy to examine daunomycin distribution in drug-resist
ant KB cells. An examination of both fixed and live cells
indicated no nuclear drug accumulation but instead a major
localization of daunomycin in Golgi and lysosomes (23). The
results described in the present study therefore suggest that
HL60/Adr cells contain a drug transport pathway which is
distinct from previously analyzed multidrug-resistant human
cells. This difference as determined by microscopic studies is
futher supported by the finding that neither brefeldin A (17,
18) or lysosomotropic agents (19) alter drug accumulation, thus
suggesting that neither the Golgi apparatus or acidic vesicles
such as lysosomes are involved in drug transport. In contrast
to these agents nigericin and to a lesser extent monensin have
been found to enhance drug accumulation and inhibit efflux.
The mechanism by which these agents inhibit drug efflux is not
known. The ability of these agents to alkalinize intracellular
vesicles (19) would not seem to account for their effect on drug
accumulation. These agents are capable, however, of altering
cytosolic pH (20, 24) and may function in this way to block the
efflux pathway. Nigericin and monensin have been found to
enhance drug accumulation and inhibit efflux in other drugresistant isolates (25, 26). Previously it has been shown that
verapamil induces an increase in drug levels in anthracyclineresistant HL60 cells (10, 13). This activity does not appear to
be related to the ability of verapamil to compete for a drugbinding site on a transporter in HL60/Adr cells (13). Attempts
to modulate Ca2+ levels with specific ionophores or chelators
have not altered drug levels. It seems to
effect of verapamil on drug accumulation
Ca2+ channel blocking activity. Recently
bafilomycin AI, an agent which selectively
be indicated that the
is not related to its
we have found that
inhibits vacuolar H+-
ATPase activity at concentrations less than 10 fiM (27), en
hances drug levels and blocks efflux in both HL60/Adr and in
HL60 cells isolated for resistance to vincristine (28). These
latter cells overexpress mdrl and contain high levels of Pglycoprotein (13, 16). Vacuolar H+-ATPases are capable of
regulating the internal pH of various membrane vesicles and
organdÃ-es (29). In view of the results obtained with various
lysosomotropic agents it would not seem that vacuolar H+ATPases function in resistance by maintaining an acidic envi
ronment in certain intracellular vesicles. Possibly the involve
ment of the enzyme in regulating the pH of other organdÃ-es
such as endoplasmic reticulum is important in drug transport.
The results obtained in the present study and those described
previously (13) suggest that movement of drug from HL60/Adr
resistant cells does not involve the participation of acidic vesi
cles or the presence of a protein which can physically bind and
transport drug. The mechanism for drug transport thus appears
to be distinct from cells containing P-glycoprotein, since drug
binding by this protein seems to be an intermediate in the efflux
pathway (7, 8).
Proteins which may contribute to drug resistance in HL60/
Adr cells have been previously described. These cells have been
found to contain a M, 150,000 plasma membrane protein
(PI 50) which represents a modified (hyperphosphorylated)
form of a protein contained in drug-sensitive cells ( 12). Recently
a second protein (M, 190,000) has been detected which is
overexpressed in HL60/Adr cells and is located primarily in
the endoplasmic reticulum (13, 16). P 190, which can bind ATP
(13), is reactive with an antibody prepared against a synthetic
peptide which corresponds to the deduced sequence of P-gly
coprotein (16). The sequence of this peptide is also contained
in HAM-2 (histocompatibility antigen modifier), a transporter
protein involved in antigen presentation (30). It is thus indicated
that P 190, P-glycoprotein, and HAM-2 share a minor sequence
homology. P 190 may thus be a member of a family of proteins
which are involved in a variety of transport processes. It would
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DRUG RESISTANCE IN HL60 CELLS
thus be expected that this protein is involved in the events
leading to the redistribution of drug from nuclear material.
REFERENCES
1. Juliano, R. L., and Ling, V. A surface glycoprotein modulating drug perme
ability in Chinese hamster ovary cell mutants. Biochim. Biophys. Acta, 455:
152-162, 1976.
2. Peterson, R. H. F.. and Biedler, J. L. Plasma membrane proteins and
glycoproteins in Chinese hamster cells sensitive and resistant to actinomycin
D. J. Supramol. Struct., 9: 289-298, 1978.
3. 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.
4. Carman, D., and Center. M. S. Alterations in surface membranes in Chinese
hamster lung cells resistant to Adriamycin. Biochem. Biophys. Res. Com
mun., 105: 157-163, 1982.
5. Inaba, M., Kobayashi, H., Sakurai, Y., and Johnson, R. K. Active efflux of
daunorubicin and Adriamycin in sensitive and resistant sublines of P388
leukemia. Cancer Res., 39: 2200-2203, 1979.
6. Skovsgaard, T. M. Mechanisms of resistance to daunorubicin in Ehrlich
asciles tumor cells. Cancer Res., 38: 1785-1791, 1978.
7. Safa, A. R., Glover, C. J., Myers, M. B., Biedler, J. L., and Felsted. R. L.
Vinblastine photoaffinity labeling of a high molecular weight surface mem
brane glycoprotein specific for multidrug-resistant cells. J. Biol. (linn.. 261:
6137-6140, 1986.
8. Cornwell, M. M., Safa, A. R.. Felsted, R. L., Gottesman. M. M., and Pastan,
1. Membrane vesicles from multidrug-resistant human cancer cells contain a
specific 150- to 170-kDa protein detected by photoaffinity labeling. Proc.
Nati. Acad. Sci. USA, 83: 3847-3850,
9. Hamada, H., and Tsuruo., T. Purification of the 170- to 180-kilodalton
membrane glycoprotein associated with multidrug resistance. J. Biol. Chem..
26 3: 1454-1458, 1988.
10. Bhalla. K., Hindenburg. A.. Taub. R. N., and Grant. S. Isolation and
characterization of an anthracycline-resistant human leukemic cell line. Can
cer Res., 45: 3657-3662, 1985.
11. Marsh, W., Sicheri, D., and Center, M. S. Isolation and characterization of
Adriamycin-resistant HL-60 cells which are not defective in the initial
intracellular accumulation of drug. Cancer Res., 46: 4053-4057, 1986.
12. Marsh, W., and Center, M. S. Adriamycin resistance in HL60 cells and
accompanying modification of a surface membrane protein contained in
drug-sensitive cells. Cancer Res., 47: 5080-5086, 1987.
13. McGrath, T., Latomi, C., Arnold, S. T., Safa, A. R., Felsted. R. L., and
Center, M. S. Mechanisms of multidrug resistance in HL60 cells: analysis of
resistance associated membrane proteins and levels of nuli gene expression.
Biochem. Pharmacol.. 38: 3611-3619. 1989.
14. Cass, C. E., Janowska-Wieczorek, A., Lynch, M. A., Sheinin, H.. Hinden
burg, A. A., and Beck, W. T. Effect of duration of exposure to verapamil on
vincristine activity against multidrug-resistant human leukemic cell lines.
Cancer Res., 49: 5798-5804, 1989.
15. McGrath. T., and Center, M. S. Mechanisms of multidrug resistance in
HL60 cells: evidence that a surface membrane protein distinct from Pglycoprotein contributes to reduced cellular accumulation of drug. Cancer
Res., 48: 3959-3963, 1988.
16. Marquardt, D., McCrone, S., and Center, M. S. Mechanisms of multidrug
resistance in III wi cells: detection of resistance-associated proteins with
antibodies against synthetic peptides that correspond to the deduced sequence
of P-glycoprotein. Cancer Res., 50: 1426-1430, 1990.
17. Fugiwara, T., Oda, K., Yokata, S.. Takatsuki. A., and Ikehara. Y. Brefeldin
A causes disassembly of the Golgi complex and accumulation of secretory
proteins in the endoplasmic reticulum. J. Biol. Chem., 263: 18545-18552,
1988.
18. Dom, R. W., RUSS,G., and Yewdell, J. W. Brefeldin A redistributes resident
and interant Golgi proteins to the endoplasmic reticulum. J. Cell Biol., 109:
61-72, 1989.
19. Ohkuma, S., and Poole, B. Fluorescence probe measurement of (he intralysosomal pH in living cells and the perturbation of pH by various agents.
Proc. Nati. Acad. Sci. USA. 75: 3327-3331, 1978.
20. Pressman, B. C. Biological applications of ionophores. Annu. Rev. Biochem.,
«: 501-530, 1976.
21. Hindenburg, A. A., Gervasoni, J. E., Krishina, S., Stewart, V. J., Rosado,
M., Lutzky, J., Bhalla, K., Baker, M. A., and Taub, R. N. Intracellular
distribution and pharmokinetics of daunorubicin in anthracycline-sensitive
and -resistant HL60 cells. Cancer Res., 49: 4607-4614, 1989.
22. Gervasoni, J. E., Jr., Fields, S. Z., Krishna, S., Baker, M. A., Rosado. M.,
Thuraisamy, K., Hindenburg, A. A., and Taub, R. N. Subcellular distribution
of daunorubicin in P-glycoprotein-positive and -negative drug-resistant cell
lines using laser-assisted confocal microscopy. Cancer Res., 51: 4955-4963,
1991.
23. Willingham, M. C, Cornwell, M. M., Cardarelli, C. O., Gotlesman, M. M.,
and Pastan, I. Single cell analysis of daunomycin uptake and efflux in
multidrug-resistant and -sensitive KB cells: effects of verapamil and other
drugs. Cancer Res., 46: 5941-5946, 1986.
24. Tartakoff, A. M. Perturbation of vesicular traffic with the carboxylic ionophorc monesin. Cell, 32: 1026-1028, 1983.
25. Klohs, W. D., and Steinkampf. R. W. Possible link between the intrinsic
drug resistance of colon tumors and a detoxification mechanism of intestinal
cells. Cancer Res., 48: 3025-3030. 1988.
26. Sehesled, M., Skovsgaard, T., and Roed. H. The carboxylic ionophore
monensin inhibits active drug efflux and modulates in vitro resistance in
daunorubicin resistant Ehrlich ascites tumor cells. Biochem. Pharmacol., 37:
3305-3310,1988.
27. Bowman, E. J., Siebers, A., and Altendorf, K. Bafilomycins: a class of
inhibitors of membrane ATPases from microorganisms, animal cells, and
plant cells. Proc. Nati. Acad. Sci. USA, «5:7972-7976.
28. Marquardt, D., and Center, M. S. Involvement of vacuolar H*-adenosine
triphosphatase activity in multidrug resistance in HL60 cells. J. Nati. Cancer
Inst., 83: 1098-1109, 1991.
29. MallÃ-n.in.L, Fuchs, R., and Hclenium. A. Acification of the endocytic and
exocytic pathways. Annu. Rev. Biochem., 55: 663-700. 1986.
30. Monaco, J. J., Cho, S., and Attaya, M. Transport protein genes in the murine
MHC: possible implications for antigen processing. Science (Washington
DC), 250: 1723-1726, 1990.
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Drug Transport Mechanisms in HL60 Cells Isolated for
Resistance to Adriamycin: Evidence for Nuclear Drug
Accumulation and Redistribution in Resistant Cells
David Marquardt and Melvin S. Center
Cancer Res 1992;52:3157-3163.
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