Download Elevated Level of Nuclear Protein Kinase C in

[CANCER RESEARCH 52. 3750-3759. July I. 1992)
Elevated Level of Nuclear Protein Kinase C in Multidrug-resistant MCF-7 Human
Breast Carcinoma Cells
Sung Ae Lee, James W. Karaszkiewicz, and Wayne B. Anderson1
Laboratory of Cellular Oncology, National Cancer Institute, NIH, Bethesda, Maryland 20892
ABSTRACT
Previous studies have demonstrated elevated levels of protein kinase
C (PKC) activity in multidrug-resistant human breast carcinoma MCF7/ADR cells compared to control drug-sensitive MCF-7/WT cells (R. L.
Fine, J. Patel, and B. A. Chabner, Proc. Nati. Acad. Sci. USA, 85:582586, 1988). In our present studies, immunohistochemical localization
analysis using a polyclonal PKC antibody recognizing the a, ¡i.and y
subtypes of PKC demonstrates that immunoreactivity is enhanced in
MCF-7/ADR cells, with pronounced staining noted in the nuclear region.
Other studies with purified nuclei isolated from MCF-7/ADR cells also
show a marked increase in the intensity of immunostaining for PKC when
compared to nuclei prepared from control MCF-7/WT cells. Western
blot analysis of proteins extracted from purified nuclear preparations
further establishes an increase in PKC enzyme protein associated with
the nuclear fraction of MCF-7/ADR cells. Subcellular fractionation stud
ies also indicate that MCF-7/ADR cells have 4-8 times higher nuclear
PKC activity compared to that of control MCF-7/WT cells. MCF-7/
ADR cells also possess 3-5-fold elevated cytosolic PKC activity, while a
<2-fold increase is found in PKC activity associated with the plasma
membrane fraction of MCF-7/ADR cells. Examination of these extracts
with PKC isotype-specific antisera, as well as by DEAE-cellulose chromatography, reveals that nuclei prepared from MCF-7/ADR cells contain
markedly elevated amounts of a slightly altered form of PKCa. These
results suggest that elevated levels of a modified form of PKCa at the
nucleus may play a role in modulating nuclear events to promote the
development of multidrug resistance in MCF-7 cells.
INTRODUCTION
nisms are (a) alterations in glutathione metabolism (8-11), (b)
a decrease in metabolic activation of drug ( 12), and (c) differ
ential oxygen free-radical susceptibility (13). It also has been
reported that PKC activity is increased in several cell types
exhibiting the MDR phenotype, including Adriamycin-resistant
MCF-7 human breast carcinoma cells (14). The enhanced levels
of PKC in MCF-7 cells resistant to Adriamycin and the mod
ulation of the drug-resistant phenotype by agents which alter
PKC activity (i.e., TPA, which first activates and then subse
quently down-regulates PKC, as well as H-7 [l-(5-isoquinolinesulfonyl)-2-methylpiperazine]
PKC inhibitor) suggest a role
for PKC in multidrug resistance (14-17). Thus, it is of impor
tance to characterize the biological and regulatory properties of
PKC found elevated in multiple drug-resistant cell types.
To better define a possible role of PKC in the modulation of
the multidrug resistance phenotype, we have assessed changes
in PKC activity, subcellular distribution, and isotype pattern in
drug-sensitive MCF-7/WT cells and in drug-resistant MCF-7/
ADR cells. The MCF-7/ADR cells were found to possess
markedly elevated levels of a modified form of PKCa in the
nucleus. These results suggest that an increase in a specific
form of PKCa in the nucleus of MCF-7 human breast carci
noma cells may play a role in modulating nuclear events such
as altered transcription to promote the development of multidrug resistance in MCF-7 cells.
MATERIALS
Development of resistance to cancer chemotherapeutic agents
such as doxorubicin (Adriamycin) and vinblastine severely lim
its the use of these drugs in cancer treatment. A particular
phenotype of resistance, called multidrug-resistance (MDR),2
exhibits a broad spectrum of resistance to anticancer drugs
derived from natural products or their derivatives including
anthracyclines, Vinca alkaloids, epipodophyllotoxins, and actinomycin D (l, 2). Although the mechanism associated with
the development of multidrug resistance is still unclear, a
common feature of multidrug resistance is a net decrease in the
intracellular accumulation of drugs as a consequence of en
hanced drug efflux and the overexpression of a membraneassociated P-glycoprotein (3-7). However, several lines of evi
dence suggest that mechanisms in addition to a net decrease in
drug accumulation may be also involved in modulating multidrug resistance. Among other possible contributing mecha-
AND METHODS
Materials. Doxorubicin (Adriamycin) and histone HI (type HIS.
lysine-rich histone) were purchased from Sigma (St. Louis, MO). Leupeptin, »pmtimn. (4-amidinophenyl)methanesulfonyl
fluoride (watersoluble form of PMSF), soybean trypsin inhibitor, pepstatin A, dithiothreitol, biotinylated goat anti-rabbit IgG, alkaline phosphatase-conjugated streptavidin, nitroblue tetrazolium chloride, and S-bromo-4chloro-3-indolyl phosphate were obtained from Boehringer-Mannheim
(Indianapolis, IN). DNase II and CHAPS detergent were from Calbiochem (La Jolla, CA). 1,2-Dioleoyl-sn-glycerol and phosphatidylserine
were from Avanti Polar Lipids (Pelham, AL). [-Y-"P]ATP was pur
chased from New England Nuclear (Boston, MA).
Cell Lines. MCF-7 human breast cancer cell lines (drug-sensitive
MCF-7/WT cells and multidrug-resistant MCF-7/ADR cells) were
generously provided by Dr. Kenneth Cowan (National Cancer Institute,
NIH, Bethesda, MD). Cells were grown in Richter's IMEM supple
mented with 2 HIM L-glutamine, 2 mg/ml L-proline, 50 Mg/m' gentamicin, and 10% fetal bovine serum (Flow Laboratories, New York, NY)
at 37°Cunder a humidified atmosphere of 5% COj in air. Cells were
passaged twice weekly and monitored routinely for their resistance to
Adriamycin. To maintain a highly drug-resistant cell population the
multidrug-resistant MCF-7/ADR cells were periodically reselected by
growing them in the presence of increasing concentrations of Adria
mycin (7-10 /JM) for approximately 2-month periods of time. Experi
ments were carried out using cells between passages 5 and 20. Another
set of independently derived drug-sensitive and drug-resistant MCF-7
cell lines was kindly provided by Dr. Grace Yeh [National Cancer
Institute; designated as MCF-7(GY) cells to distinguish from the MCF7 cell lines obtained from Dr. Kenneth Cowan). Drug-sensitive MCF7(GY)/WT cells and multidrug-resistant MCF-7(GY)/W0.3 and MCF7(GY)/WIO cells were grown in RPMI 1640 (GIBCO, Gaithersburg,
3750
Received 3/3/92; accepted 4/23/92.
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.
1To whom requests for reprints should be addressed, at Laboratory of Cellular
Oncology, National Cancer Institute, NIH, Bldg. 36. Room 1D22, Bethesda, MD
20892.
2The abbreviations used are: MDR, multidrug resistance; PKC. protein kinase
C; TPA. ^-O-tetradecanoylphorbol-U-acetate;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio)propanesulfonate;
MTT. 3-(4.5-dimethyl(thiazol-2-yl)3.5-diphery|tetradium bromide; EGTA, ethyleneglycol-bisW-aminoethyl ether)A/,A/,.V'..\"-tetraaceticacid; D-PBS. Dulbecco's phosphate buffered saline; IMEM,
improved minimum essential medium; PBS, phosphate-buffered saline; BSA,
bovine serum albumin; SDS. sodium dodecyl sulfate; IC50, drug concentration
required for SO1";cytotoxicity: CM-free. Ca2*- and Mg2*-free.
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1992 American Association for Cancer Research.
ELEVATED NUCLEAR PKC AND DRUG RESISTANCE
MD) supplemented with 2 HIML-glutamine and 10% fetal bovine serum
(Flow Laboratories) at 37°Cunder a humidified atmosphere of 5% CO2
in air.
PKC Antibodies. Four different polyclonal PKC antibodies were used
in these studies. Antibodies were raised in rabbits against keyhole limpet
hemocyanin-conjugated synthetic peptides corresponding (a) to the
NH2-terminal pseudosubstrate region common to isotypes a, ß,
and y
(FARKGALRQKNV) and thus pan-specific for these isotypes, (b) to
the a isotype-specific hinge region peptide (KLGPAGNKVISPSED),
(c) to the ß
isotype-specific COOH-terminal region (SEFEGFSFVNSEFLKPEVKS), and (d) to the 7 isotype-specific COOH-terminal re
gion (AEFQGFTYVNPDFVH)
of PKC. The isotype specificity was
screened using extracts prepared from COS-7 cells transfected with the
«,ß,and 7 isotypes of PKC. Each antiserum was found to be specific
for the PKC isotype indicated and exhibited no cross-reactivity with
the other PKC isotypes tested.
Immunohistochemical Localization of PKC. MCF-7/WT cells and
MCF-7/ADR cells grown on Lab-Tek Permanox tissue culture chamber
slides were washed with PBS, fixed with 4% formaldehyde/0.05%
glutaraldehyde in PBS, and then permeabilized with 0.1% Triton X100 in PBS. The permeabilized cells were sequentially incubated with
10% normal horse serum, 10% normal goat serum, and 2% BSA
(globulin-free) in PBS for 30 min each to block nonspecific IgG binding.
Then, the cells were incubated overnight at 4°Cwith PKC antiserum
(pan-specific for a, ß,and 7 isotypes of PKC) in PBS containing 2%
normal goat serum, 1% BSA (globulin-free), and 0.05% Triton X-100.
The cells then were rinsed with PBS and subsequently incubated with
biotinylated goat anti-rabbit IgG in PBS containing 1% BSA (globulinfree) and 0.01 % Tween 20. The immunoreactivity to PKC was localized
by incubation with alkaline phosphatase-conjugated avidin D and sub
sequent color development by incubation with Vector ABC kit (Vector
Laboratories, Burlingame, CA).
Preparation of Nuclear-Cytoskeletal Components. Intact MCF-7/WT
cells and MCF-7/ADR cells grown on Lab-Tek Permanox tissue culture
chamber slides were rapidly washed with warm (30°C)PBS and ex
tracted for 10-14 min at 30°Cby incubation with buffer solution
containing 0.1 M piperazine-A',Ar'-bis(2-ethanesulfonic acid) (pH 6.9),
sample was 0.4 mM for each. Phosphatidylserine (25 /jg) and diolein
(2.5 Mg)were added to some tubes in a volume of 20 n\ to demonstrate
phospholipid-dependent protein kinase activity. After incubation for
6-10 min at 30°C,50 n\ of aliquots of the reaction mixture were spotted
onto paper strips (Whatman P-81 ion exchange chromatography pa
per), and the strips were immediately immersed into 75 mM phosphoric
acid. The P-81 papers were washed at least 4 times with 75 mM
phosphoric acid and air dried, and radioactivity retained on the papers
was determined. Protein kinase C activity was calculated from the
difference in 32Pincorporated into histone in the presence and absence
of added phospholipids and calcium and was expressed as '2P incorporated/min/mg protein.
Colorimetrie MTT Test for Cytotoxicity to Adriamycin. MCF-7 cells
were seeded in 24-well plates (Costar, Cambridge, MA) at a density of
approximately 25,000 cells/well in 1MEM as described above. After 24
h the medium was removed and I nil aliquots of IMl1'M containing
various concentrations of doxorubicin and supplemented with 2 mM Lglutamine, 2 mg/ml i.-proline, 50 pg/ml gentamicin and 10% heatinactivated fetal bovine serum were added to each well. After 48 h of
incubation at 37°C,100 nl of MTT at a concentration of 5 mg/ml in
D-PBS were added to each well, and the cultures were further incubated
for 2 h at 37°C.The reaction was terminated by removal of the medium
along with the MTT dye, and the formazan precipitate formed was
dissolved in 1 ml of dimethyl sulfoxide. The relative number of viable
cells remaining after Adriamycin treatment was estimated by the change
in absorbance at 590 nm. The relative percentage of surviving cells was
calculated asAbsorbance in wells treated with doxorubicin + Absorb
ance in control wells without doxorubicin x 100
Western Blot Analysis of PKC. Subconfluent MCF-7 cells grown in
IMEM were rapidly washed twice with ice-cold Ca:*- and Mg;+-free
(CM-free) D-PBS. A hot SDS total cell extract then was prepared by
immediately scraping the cells into boiling SDS-polyacrylamide gel
electrophoresis sample buffer and drawing through a 23-gauge needle
10 times to shear DNA (19). The extracts were then boiled for 5 min.
Electrophoresis of the extracted proteins was carried out on 8% SDSpolyacrylamide gel electrophoresis minigels unless otherwise described.
The protein bands then were transferred electrophoretically to Immobilon-P membrane (Millipore, Bedford, MA). Following electrotransfer, the membrane was incubated with 2% (w/v) Carnation nonfat dry
milk in CM-free D-PBS for 30 min at room temperature to block
nonspecific IgG binding sites and then washed with CM-free D-PBS
for 10 min. The membrane then was incubated overnight at 4°Cwith
protein kinase C antiserum diluted 1:500 in CM-free D-PBS containing
2% nonfat dry milk. The membrane exposed to antiserum then was
washed sequentially with 0.25% Tween 20 in CM-free D-PBS for 10
min, with 0.05% Tween 20 in CM-free D-PBS twice (10 min each
wash), and finally with CM-free D-PBS for 10 min at room tempera
ture. After primary antiserum incubation and washings, the membrane
was incubated with biotinylated secondary antibody diluted 1:1000 in
CM-free D-PBS containing 2% nonfat dry milk for 1.5-2.5 h at room
temperature and then washed as described above. The washed mem
brane then was incubated with alkaline phosphatase-conjugated streptavidin diluted 1:1000 in CM-free D-PBS containing 2% nonfat dry
milk for 30 min at room temperature and again washed as described
above. PKC was visualized by incubation with 0.4 mM nitroblue tetrazolium-0.4mM 5-bromo-4-chloro-3-indolyl-phosphatein
100 mM TrisCl (pH 9.5) buffer solution containing 100 mM NaCl and 10 mM MgCl2
at room temperature for 10-20 min. To terminate the color develop
ment reaction the membrane was washed with 50 mM EDTA, subse
quently washed with deionized 11 (). and then air dried.
Protein Determination and Data Expression. Protein concentration
was determined by Bradford protein assay (20) using BSA as standard.
Data are expressed as mean ±SEM. Error bars smaller than the
symbols or lines used in the figures have been omitted.
5 mM MgCb, 0.2 mM EGTA, 4 M glycerol, 100 ng/m\ leupeptin, and
0.1% Triton X-100. The solubilized fraction was quickly removed to
leave nuclear-cytoskeletal structures attached to the substratum. The
remaining nuclei and cytoskeletal structures were washed twice with
cold PBS and subsequently processed for the immunohistochemical
localization of PKC as described above.
Subcellular Fractionation. Cells were washed twice with ice-cold Ca2+and Mg:*-free D-PBS and then harvested by scraping from culture
dishes into ice-cold Equilibration Buffer [20 mivi Tris-HCl (pH 7.5)
containing 2 m\i EDTA, 2 mM EGTA, 2 mM dithiothreitol, and
protease inhibitors (100 ;ig/ml leupeptin, 50 iig/ml aprotinin, 50 p%/
ml 4-amidinophenyl-methanesulfonyl fluoride, 10 iig/ml soybean trypsin inhibitor, and 100 ng/ml pepstatin A)], and then disrupted by 100
strokes (pestle B) with a Dounce homogenizer. Crude nuclei were
isolated by centrifugation of total cell homogenate at 1000 x g for 5
min. This crude nuclear pellet was subsequently purified by sucrose
density centrifugation as described previously (18). The purity of the
nuclear preparations was determined by electron microscopy and was
routinely monitored by phase contrast microscopy after staining with
méthylène
blue. The plasma membrane-enriched fraction was prepared
by centrifugation of the 1,000 x g supernatant at 10,000 x g for 20
min. The cytosolic fraction was isolated by centrifugation of the 10,000
x g supernatant at 100,000 x g for 60 min. For PKC activity measure
ment the total cell homogenate, purified nuclear fraction, and plasma
membrane-rich fractions were solubilized with 1% (w/v) CHAPS on
ice overnight.
Assay of PKC Activity. The reaction was initiated by addition of 13 ng of protein sample to a reaction mixture containing 20 mM Tris-Cl
(pH 7.5), 1 mM CaCl2, 10 mM MgCl2, 100 /¿M
[7-"P]ATP (3 x 10"
RESULTS
cpm), 2 mM dithiothreitol, and 200 pg/m\ histone type III-S or 4 ¿IM
PKC substrate peptide [(Ser2i)PKC( 19-31)] (GIBCO) as phosphoacImmunohistochemical Localization of PKC in MCF-7/WT and
localization of
ceptor substrate in a total volume of 100 n\. The final concentration of MCF-7/ADR Cells. The immunohistochemical
PKC was determined with rabbit anti-peptide antiserum panEDTA and EGTA in the reaction mixture contributed by the protein
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ELEVATED NUCLEAR PKC AND DRUG RESISTANCE
specific for the o, ß,and 7 isotypes of PKC prepared as
described in "Materials and Methods." In intact MCF-7 cells,
multidrug-resistant MCF-7/ADR cells showed a markedly en
hanced immunoreactivity to PKC compared to that of drugsensitive MCF-7/WT cells (Fig. 1). The most pronounced
difference was found in the nuclear region. A dense immunostaining was found in the nuclear region of MCF-7/ADR cells
while little immunostaining was present in the perinuclear
region of MCF-7/WT cells. This finding was confirmed in
studies using isolated nuclei (Fig. 2). Nuclei isolated from MCF7/ADR cells showed a marked increase in intensity of immuno
staining for PKC compared to nuclei prepared from drugsensitive MCF-7/WT cells. A similar result was observed with
a isotype-specific PKC antibody, while no significant difference
in the intensity of immunostaining for ß-isotypePKC was found
in MCF-7/WT and MCF-7/ADR cells (data not shown).
Subcellular Distribution of PKC Activity in MCF-7/WT Cells
and MCF-7/ADR Cells. It has been previously demonstrated
that PKC activity is elevated in MCF-7/ADR cells compared
to MCF-7/WT cells (14). To better characterize the elevated
PKC activity found in MCF-7/ADR cells, the subcellular dis
tribution of PKC activity in MCF-7/WT and MCF-7/ADR
cells was compared (Table 1). In the process of subcellular
traci¡on;iiion. the nuclear pellets isolated from MCF-7/WT and
MCF-7/ADR cells were further purified as described by
Thomas et al. (18). The purity of the nuclear fraction was
verified by electron microscopic examination (data not shown).
The total cell homogenate of MCF-7/ADR cells exhibited
about a 4-fold increase in total PKC activity compared to that
of MCF-7/WT cells. The most striking difference in total PKC
activity, and also in specific activity, was found in the nuclear
fraction. The nuclear fraction isolated from MCF-7/ADR cells
exhibited a 4-8-fold higher PKC specific activity when com
pared to those of MCF-7/WT cells. Similar results were ob
served when the nuclear preparation was isolated following
disruption of the cells in homogenization buffer containing
detergent such as 0.05% Triton X-100 or containing 0.5 M
hexylene glycol (data not shown). A higher level (3-5-fold) of
PKC activity was also found in the cytosolic fraction of MCF7/ADR cells, while a lesser increase (<2-fold) was noted in
plasma membrane-associated PKC activity of MCF-7/ADR
cells. To exclude the possibility that the significant pool of
nuclear PKC activity found in the MCF-7/ADR cells might
originate from residual contamination from the cytosolic pool,
we carried out a time course analysis of changes in the distri
bution and down-regulation of PKC in the different subcellular
fractions in response to treatment of these cells with 1 ^M TPA
(Fig. 3). Exposure of MCF-7/ADR cells to l /tM TPA resulted
MRS
Control
PKC
Aby
MCF-7/ADR
MCF-7/WT
Fig. 1. Immunohistochemical localization of protein kinase C in intact drug-sensitive MCF-7/WT and multidrug-resistant MCF-7/ADR human breast carcinoma
cells. MCF-7/WT and MCF-7/ADR cells were fixed and analyzed for PKC by immunohistochemical staining as described in "Materials and Methods." Primary
antibody used was rabbit anti-peptide antiserum (pan-specific for MHz-terminal pseudosubstrate region of a. ti, and y isotype of PKC). In the control group, normal
rabbit serum was used in place of PKC antibody.
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ELEVATED NUCLEAR PKC AND DRUG RESISTANCE
MRS
Control
PKC
Aby
MCF-7/WT
MCF-7/ADR
Fig. 2. Immunohistochemical localization of protein kinase C in the nuclear-cytoskeletal fraction of drug-sensitive MCF-7/WT and multidrug-resistant MCF-7/
ADR human breast carcinoma cells. The nuclear-cytoskeletal fraction was prepared as described in "Materials and Methods." Rabbit anti-peptide antiserum (panspecific for NHj-terminal pseudosubstrate region of «,/i, and y isotypes of PKC) was used in this study, while normal rabbit serum was used as a control. The
immunoreactivity to PKC visualized with a Vector ABC kit was photographed with an Olympus Vanox microscope at x 400.
Table 1 Subcellular distribution of protein kinase C activity in control drug-sensitive MCF-7/ WT and multidrug-resistant MCF-71 ADR cells
Subcellular fractionation and PKC activity measurement were carried out as described in "Materials and Methods." Data are expressed as mean ±SEM.
Protein kinase C activity
units
(nmol/min/108
cells)MCF-7/ADR32.51protein)Fold
Subcellular
fractionTotal
homogenate
Nucleus
Plasma membrane
CytosolMCF-7/WT8.79
±1.37
0.78 ±0.08
1.21 ±0.08
7.28 ±0.64Total
±2.03
5.75 ±0.35
2.66 ±0.37
34.84 ±4.23Specific
in a rapid loss of PKC activity from the cytosolic fraction within
30 min (to ~20% of control). Concomitantly, there was a
pronounced increase in plasma membrane-associated PKC ac
tivity (to ~370% of control). In contrast, the nuclear pool of
PKC activity was not appreciably altered at early (30- and 90min) time periods following exposure of the cells to TPA,
although a rise (to ~160% of control) in nuclear PKC activity
was noted at 3 h after the addition of TPA. Activity analysis
also revealed that the time course and extent of down-regulation
of PKC in the different subcellular fractions in response to
prolonged exposure to TPA also are different. Cytosolic PKC
was translocated and diminished within 30 min of exposure to
TPA, although a residual (~20% of control) level of cytosolic
activity
(nmol/min/mg
increase3.7
increase4.5
±0.04
±0.07
7.4
0.27 ±0.03
1.37 ±0.08
0.89 ±0.04
1.30 ±0.03
2.2
4.8MCF-7/WT0.250.46 ±0.04MCF-7/ADR1.12
2.18 ±0.26Fold
5.1
1.5
4.7
PKC activity remained throughout the 24-h period of TPA
treatment. The dramatic increase in plasma membrane-associ
ated PKC was transient, and within 90 min after TPA treatment
the PKC activity in the plasma membrane fraction had fallen
by 60%. The plasma membrane PKC activity continued to show
a drop through the remainder of the 24-h treatment period, but
with only a maximal decrease to 40% of control. The nuclear
pool of PKC, however, was completely down-regulated within
12 h of TPA treatment, and no PKC activity could be detected
in the nuclear fraction at either the 12- or 24-h time periods.
The observed differences in the time course and extent of TPAinduced redistribution and down regulation of PKC associated
with the cytosolic, plasma membrane, and nuclear subcellular
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ELEVATED NUCLEAR PKC AND DRUG RESISTANCE
the subsequent detergent-solubilized nuclear PKC activity in
both drug-sensitive MCF-7/WT cells and multidrug-resistant
MCF-7/ADR cells. These results indicate that the majority of
PKC remains with the insoluble nuclear fraction and can be
removed by detergent.
Correlation between Nuclear PKC Activity and Drug Resist
ance. The profound increase in nuclear PKC activity observed
in multidrug-resistant MCF-7/ADR cells led us to investigate
whether the level of nuclear PKC activity might correlate with
the degree of drug resistance in MCF-7 cells. For this study,
we used three different MCF-7 cell lines with different levels of
drug resistance: one control MCF-7/WT cell line; and two
drug-resistant MCF-7/ADR cell lines designated as MCF-7/
ADRC| cells and MCF-7/ADRjm cells. The relative drug resist
ance of the three MCF-7 cell lines was determined by comparing
the chemosensitivity of these cells to the cytotoxic effect of
doxorubicin (Adriamycin) as determined by the MTT colori
metrie assay as described in "Materials and Methods." Under
the given culture and assay conditions, MCF-7/ADRjm cells
12
18
24
TIME (hours)
Fig. 3. Effect of TP A treatment of MCF-7/ADR cells on the distribution and
down-regulation of protein kinase C activity found in the cytosolic, plasma
membrane, and nuclear subcellular fractions. MCF-7/ADR cells were treated
with l /JM TPA for the indicated periods of time, the cells were harvested by
scraping, and cytosolic, plasma membrane, and nuclear fractions were prepared
as described in "Materials and Methods." Assays for PKC activity present in the
subcellular fractions were carried out as described in "Materials and Methods"
using PKC substrate peptide [(Ser")PKC( 19-31)] (GIBCO) as phosphoacceptor
substrate. Purified nuclei and cytosol were prepared as described in "Materials
and Methods."
Table 2 Detergent-solubilized PKC activity remaining in nuclei isolated from
MCF-7/WTand MCF-7/ADR cells following either high salt concentration
washing or DNase II digestion of the nuclear preparations
Purified nuclei isolated from MCF-7/WT and MCF-7/ADR cells were sus
pended in EB" alone, in EB containing 0.5 M NaCl, or in EB containing 0.4 mg/
ml DNase II for 1 h. These suspensions were centrifuged at 1000 x g for 10 min
to obtain the extracted nuclear pellets, and these nuclear pellets then were
resuspended in buffer containing 1% CHAPS detergent to solubilize remaining
nuclear PKC. The detergent-solubilized nuclear fractions were assayed for PKC
activity as described in "Materials and Methods" using PKC substrate peptide
[(Ser")PKC( 19-31)1 (GIBCO) as phosphoacceptor substrate. The detergent-sol
ubilized PKC activities found in the nuclei pretreated as indicated are given as
nmol "P incorporated/min/mg protein. Data are expressed as mean ±SEM.
Detergent-solubilized
Pretreatment with
EB" alone
were the most resistant with a IC50 of approximately 3 /¿M,
while MCF-7/ADRcf cells exhibited a resistance (IC50 -0.3 ,;M)
intermediate between MCF-7/ADRjm cells and MCF-7/WT
cells (Fig. 4; Table 3). Control, drug-sensitive MCF-7/WT cells
exhibited a 1C?,]of 0.04 /UMAdriamycin.
The nuclear PKC activity of these three cell lines was deter
mined, and the relationship between the nuclear PKC activity
and the relative drug resistance was examined (Table 3). The
100
CO
_l
90
O
80
LU
Ü
Z
70
CO
60
I-
50
nuclear protein kinase C activity
Pretreatment with
EB containing
0.5 M NaCl
Pretreatment with
EB containing
DNase II
Cell type
LLJ
MCF-7/WT
0.171 ±0.013
0.106 ±0.023
0.100 ±0.009
Ü
MCF-7/ADR
DC
1.175 ±0.064
1.728 ±0.046
1.244 ±0.059
LU
°EB. equilibration buffer [20 mM Tris-Cl (pH 7.5), 2 mvi EDTA, 2 mM EGTA,
OL
2 mM dithiothreitol, and protease inhibitors (100 nu ml leupeptin, 50 «<g/ml
III
aprotinin, 50 *ig/ml (4-amidinophenyl)methanesulfonyl
fluoride, 10 »ig/mlsoy
bean trypsin inhibitor, and 100 ng/ml pepstatin A)].
ADR
|m
MCF-7/WT
40
30
20
fractions strongly suggest that the nuclear pool of PKC found
in MCF-7/ADR cells is distinct from the cytosol and plasma
membrane pools of PKC in these cells.
Extraction of PKC from Isolated Nuclei. To determine
whether the high level of PKC activity present in the purified
nuclear fraction isolated from MCF-7/ADR cells was extractable by high salt concentration or by DNase treatment, the
purified nuclei were first washed with 0.5 M NaCl or treated
with 0.4 mg/ml DNase II prior to solubilization with detergent
(1% CHAPS) (Table 2). Prior extraction of isolated nuclei with
buffer containing either 0.5 M NaCl or 0.4 mg/ml DNase II
caused approximately a 40 and a 30% decrease, respectively, in
LU
OC
10
0
0.001 0.01 0.1
1.0
10
100
ADRIAMYCIN
Fig. 4. Relative sensitivity of control MCF-7/WT cells and multidrug-resistant
MCF-7/ADRcf and MCF-7/ADR'1" cells to the effect of 48-h treatment with
increasing concentrations of doxorubicin (Adriamycin). Chemosensitivity of drugsensitive MCF-7/WT cells and multidrug-resistant MCF-7/ADRcf and MCF-7/
ADR'"1 cells to the cytotoxic effect of doxorubicin (Adriamycin) was determined
by MTT colorimetrie assay as described in "Materials and Methods." Cells were
treated with the indicated concentrations of doxorubicin (Adriamycin) for 48 h.
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ELEVATED NUCLEAR PKC AND DRUG RESISTANCE
trast, MCF-7/ADR cells contained high levels of nuclear PKC
activity which eluted in two major peaks from the DEAE
column. The first peak eluted with 140 mM NaCl, while the
second, more prominent, peak of activity eluted with 180 mM
NaCl. Both peaks of nuclear PKC activity noted with the MCFProtein kinase C activity*
7/ADR extract were found to be immunoreactive with both the
(nmol 32P incorporated/
pan-specific PKC antiserum and with PKC«-specific antiserum
protein)Cell
min/mg
to
by dot blot analysis (data not shown).
Adriamycin resistance to
type"MCF-7/W
(IC,o
(MM)0.04 Adriamycinc160600 Western Blot Analysis of PKC in MCF-7/WT and MCF-7/
homogenate0.25
ADR cells. Our finding that the increase in immunohistochem±0.04
±0.03
MCF-7/ADR''
ical staining and in PKC activity noted in MCF-7/ADR cells is
1.02 ±0.21 0.60 ±0.05
0.25
MCF-7/ADR""
2.82Relative
1.12 ±0.07 1.37 ±0.08
due to an increase in PKC enzyme was further established by
MCF-7(GY)/WT
1.07 ±0.10 0.18 ±0.06
Western blot analysis of total cell extracts using a PKC antibody
MCF-7(GY)/W0.3
2.70 ±0.09 1.56 ±0.09
MCF-7(GY)/W10Total 2.68 + 0.12Nucleus0.27
pan-specific for PKC isotypes «,ß,and y (Fig. 6). Markedly
3.1 5 ±0.08Sensitivity
" MCF-7/WT. MCF-7/ADRcf. and MCF-7/ADRjm cells were provided by Dr.
elevated levels of PKC protein were observed in MCF-7/ADR
Kenneth Cowan, National Cancer Institute. NIH. MCF-7(GY)/WT, MCFcells. We also examined the PKC isotype pattern present in the
7(GY)/W0.3, and MCF-7(GY)/W10 cells were provided by Dr. Grace Yen,
total cell extracts prepared from these cell lines with a, ß,and
National Cancer Institute, NIH.
* For PKC activity measurements, histone HI (type III-S) was used as substrate
y isotype-specific PKC antibodies (Fig. 6). The antibodies used
for MCF-7 cells received from Dr. Kenneth Cowan, and PKC substrate peptide
were raised in rabbits against hemocyanin-conjugated synthetic
[(Ser")PKC( 19-31)][(Gibco) was used as substrate for the (GY) series of cells.
peptides as described in "Materials and Methods." a-isotype
' Data were provided by Dr. Grace Yeh.
PKC (PKC«) and 0-isotype PKC (PKC/3) were found to be
present in the MCF-7 cell lines. MCF-7/ADR cells contained
most resistant cell line (MCF-7/ADRJM1) had the highest nuclear
markedly increased amounts of PKCa compared to that of
PKC activity among these three cell lines, while the less drugMCF-7/WT cells. No significant difference in the amounts of
resistant MCF-7/ADRcl cell line showed an intermediate nu
PKC/3 was found in MCF-7/WT cells and MCF-7/ADR cells,
clear PKC activity. The control, drug-sensitive MCF-7/WT
and we were unable to detect PKC-y in these cell lines. Recently,
cells had the lowest nuclear PKC activity. It was of interest to
additional members of the PKC family (i.e., 5, t, and f subtypes)
note that the level of nuclear PKC activity showed a correlation
have been identified (21,22). Preliminary evidence with antisera
with the degree of relative drug resistance, while the PKC
specific to the <5,e, and f isotypes of PKC indicates that none
activities found in the total homogenates were equally elevated
of these isoforms is elevated in MCF-7/ADR cells. Because of
in the two drug-resistant cell lines (MCF-7/ADRtf and MCFthe different antibody preparations used, it is not possible to
7/ADRjm) compared to that of the drug-sensitive MCF-7/WT
Table 3 Relationship between nuclear protein kinase C activity and sensitivity to
the cytotoxic effect of Adriamycin in drug-sensitive MCF-7/WT and multidrugresistant MCF- 7/A DR cells
Chemosensitivity to the cytotoxic effect of Adriamycin (48-h treatment) was
determined by the MTT colorimetrie assay as described in "Materials and
Methods." Data are expressed as mean ±SEM.
cells.
To further establish the relationship between nuclear PKC
activity and relative drug resistance in MCF-7 cell lines, we
determined the PKC activities of various subcellular fractions
prepared from a second set of independently isolated drugsensitive and drug-resistant cells derived from additional MCF7 cell lines [MCF-7(GY) cell lines, kindly provided by Dr.
Grace Yeh]. Again, a good correlation was noted between the
level of nuclear PKC activity and the relative degree of drug
resistance exhibited by these MCF-7(GY) cell lines (Table 3).
Although PKC activities associated with the plasma membrane
and cytosolic fractions were also somewhat elevated in these
drug-resistant cell lines, a close relationship between changes
in cytosolic and plasma membrane PKC activity and relative
drug resistance was not evident.3
DEAE-Cellulose Chromatography of Nuclear PKC Activity
from MCF-7/WT and MCF-7/ADR Cells. To further character
ize the elevated nuclear PKC activity found in MCF-7/ADR
cells, we have compared the DEAE-cellulose chromatography
elution profiles of the detergent-solubilized PKC activities ob
tained from nuclei isolated from MCF-7/WT and MCF-7/ADR
cells (Fig. 5). The elution profile of nuclear PKC activity from
MCF-7/ADR cells was quite different not only quantitatively,
but also qualitatively, from that of MCF-7/WT cells. In MCF7/WT cells, the nuclear PKC activity was very low and was
mainly eluted from the column with 140 mM NaCl. Since the
level of PKC activity present in the nuclear extract from MCF7/WT cells was very low, this observation was confirmed from
a similar elution profile obtained by applying three times the
amount of nuclear protein on the column (Fig. 5, O). In con
' Unpublished data.
so
.2
o
70
> E
60
OÕ
50
Si
g S
40
Z(N
£„
30
o
°"x
a
-B
20
10
8
12
16
24
0.0
FRACTION NUMBER
Fig. 5. DEAE-cellulose column elution profile of protein kinase C activity
detergent-solubilized from nuclei isolated from MCF-7/WT and MCF-7/ADR
cells. Three hundred ng of nuclear protein detergent-solubilized (1% CHAPS)
from nuclei isolated from MCF-7/WT (•)and MCF-7/ADR (A) cells were
applied to a 2-ml bed volume DEAE-cellulose column equilibrated with equili
bration buffer [20 mM Tris-Cl (pH 7.5), 2 mM EDTA, 2 mM EGTA, 2 mM
dithiothreitol. and protein inhibitors (100 ,<u ml leupeptin, 50 Mg/ml apretinen.
50 Mg/ml (4-amidinophenylmethanesulfonyl
flouride. 10 ¿igsoybean trypsin in
hibitor, and 100 ng/ml pepslatin A)) and eluted with a linear gradient (0-0.5 M)
of NaCl in the same buffer. To facilitate better detection of the low levels of
nuclear PKC activity present in MCF-7/WT cells, DEAE-cellulose chromatog
raphy was also carried out with a 3-fold greater (900 ^g) amount of MCF-7/WT
detergent-solubilized nuclear protein (O). Five hundred-fil fractions were col
lected, and PKC activity was determined in 20-pl aliquots of each fraction as
described in "Materials and Methods." Dotted line, NaCl gradient.
3755
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ELEVATED NUCLEAR PKC AND DRUG RESISTANCE
Pan PKC Aby
Anti-PKCa
Anti-PKC^
Anti-PKC-
200KFig. 6. Western blot analysis of protein kinase C in total cell extracts of MCF-7/WT and
MCF-7/ADR cells. Electrophoresis of total cell
extracts prepared from MCF-7/WT and MCF7/ADR cells was carried out on S% SDS-polyacrylamide gel electrophoresis mini-gels as de
scribed in "Materials and Methods." Following
electrophoresis, proteins were transferred to Immobilon-P membrane. The isotype pattern of
PKC was determined by immunoblotting with
polyclonal antibodies specific for the a.. '. and y
isotypes of PKC. The antibodies used were raised
in rabbits against hemocyanin-conjugated syn
thetic peptides as described in "Materials and
Methods." Protein bands were detected with biotinylated goat anti-rabbit IgG and alkaline phosphatase-conjugated streptavidin. Arrowhead, M,
80.000 PKC band.
116K97K66K-
45K-
make a comparison between the amount of PKC«relative to
PKQ3 that might be present in the MCF-7 cell lines based upon
the intensity of the bands. Results suggest that the pan-specific
PKC antisera used recognize the PKC (apparently PKC«)pres
ent in the MCF-7/ADR cells to a much greater degree than do
the o-specific PKC antisera.
Similar immunoblot patterns were observed with purified
nuclei isolated from MCF-7/WT and MCF-7/ADR cells (Fig.
7). The purified nuclei isolated from MCF-7/ADR cells again
revealed a marked increase in immunostaining with pan-specific
PKC antibody and also contained elevated amounts of PKCa.
As in the immunoblot analysis of total cell extract, there was
little change in PKCß,and we could not detect PKC> in the
nuclei prepared from these cell lines (data not shown).
When the CHAPS-solubilized nuclear protein fractions were
electrophoresed on 10-20% SDS-polyacrylamide gradient gels,
electrophoretically transferred to Immobilon-P membranes,
and analyzed by immunoblotting with the PKC antiserum panspecific for PKC isotypes a, ß,
and 7, the MCF-7/ADR nuclear
extract revealed not only a marked increase in immunoreactivity
but also a distinct difference in the immunostaining pattern (3
bands) when compared to that of MCF-7/WT cells (2 bands)
(Fig. 8). The upper, major PKC band present in nuclei isolated
from MCF-7/ADR cells was not detectable in nuclei isolated
from MCF-7/WT cells. None of these bands was detected when
immunoblots were performed with preimmune serum (data not
shown). The reason for the difference in the pattern of PKC
bands detected by Western blot analysis of solubili/ed nuclear
proteins electrophoresed on 8% polyacrylamide gels (Fig. 7)
compared to the immunostaining pattern noted with 10-20%
polyacrylamide gradient gels (Fig. 8) is not readily apparent.
Nonetheless, this result does further suggest that MCF-7/ADR
cells do contain elevated levels of an apparently modified form
of nuclear PKC.
Pan PKC Aby
Anti-PKC«
116K97K66K-
45K-
Fig. 7. Western blot analysis of protein kinase C in nuclei isolated from MCF7/WT and MCF-7/ADR cells. The purified nuclear pellet obtained from MCF7/WT and MCF-7/ADR cells was solubilized in boiling SDS-polyacrylamide gel
electrophoresis sample buffer and drawn through a 23-gauge needle to shear
DNA. Equivalent amounts (10 ^g) of nuclear protein were electrophoresed by 8%
SDS-polyacrylamide gel electrophoresis and transferred to Immobilon-P mem
brane. PKC proteins were detected by probing the blot with rabbit anti-peptide
antiserum pan-specific for i>,/i, and y isotypes of PKC, or with rabbit anti-peptide
antiserum specific for a isotype of PKC only. Arrowhead, position of PKC band.
3756
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ELEVATED NUCLEAR PKC AND DRUG RESISTANCE
97.4K66.5K -
PKC
45.7K -
31.0K21.5K14.4K-
Fig. 8. Western blot analysis of PKC immunoreactive proteins extracted from
nuclei isolated from MCF-7/WT
and MCF-7/ADR
cells and electrophoresed
using 10-20% SDS-polyacrylamide gradient gels. Detergent (1% CHAPS)-solubilized protein fractions were prepared from purified nuclei isolated from MCF7/WT and MCF-7/ADR
cells, and the solubilized protein fractions were dena
tured in SDS sample buffer. Equivalent amounts (2 ng) of SDS denatured nuclear
protein were electrophoresed using 10-20% SDS-polyacrylamide gradient gels
and the protein bands then were electrophoretically transferred to Immobilon-P
membranes. PKC immunoreactive proteins were detected by probing the blot
with rabbit anti-peptide antiserum pan-specific for a, ß,and y isotypes of PKC.
DISCUSSION
Several lines of evidence indicate the possible involvement of
PKC in modulating cellular resistance to antitumor drugs of
the natural products class. Protein kinase C activity has been
reported to be elevated in several cell types exhibiting the
multidrug-resistance
phenotype, including MCF-7 human
breast carcinoma cells (14), HL-60 leukemia cells (23, 24),
murine fibrosarcoma cells (15), and KB human carcinoma cells
(25). Results of cell growth and cytotoxicity studies in which
PKC activity has been altered with TP A and with the H-7 [1(5-isoquinolinesulfonyl)-2-methylpiperazine]
inhibitor also in
dicate a close relationship between PKC activity and cell sen
sitivity to the cytotoxic effects of these anticancer drugs (1417). Furthermore, studies suggest that PKC-activation may be
involved in the phosphorylation of the membrane P-glycoprotein involved in drug transport. Enhanced phosphorylation of
P-glycoprotein has been noted with phorbol ester treatment of
K562/ADM cells (26), and Chambers et al. (25) have reported
that the P-glycoprotein from drug-resistant human KB carci
noma cells can serve as a good substrate for PKC in vitro.
In this study, we have presented a further characterization of
the enhanced levels of PKC found in multidrug-resistant MCF7 human breast carcinoma cells when compared to that of
control drug-sensitive MCF-7/WT cells. Our results indicate
that MCF-7/ADR drug-resistant cells contain markedly ele
vated levels of nuclear PKC. This conclusion is based upon the
following data: (a) immunohistochemical localization studies
using a PKC antibody pan-specific for a, ß,and 7 isotypes of
PKC indicated that immunoreactivity was enhanced in MCF7/ADR cells compared to that of MCF-7/WT cells, with pro
nounced staining noted in the nuclear region; (b) other immu
nohistochemical studies with nuclei isolated from MCF-7 cells
also revealed a significant increase in the intensity of iminium
staining for PKC in nuclei isolated from MCF-7/WT cells; (c)
MCF-7/ADR cells contained a markedly elevated level of nu
clear PKC activity compared to that of MCF-7/WT cells; (d)
another set of multidrug-resistant MCF-7 cell lines [MCF7(GY)/W0.3 and MCF-7(GY)/W10 cells] also was found to
exhibit an increased level of nuclear PKC activity compared to
that of control drug-sensitive MCF-7(G Y)/WT cells; (e) West
ern blot analysis of total cell extracts with a pan-specific PKC
antibody recognizing the a, 0, and y isotypes of PKC, as well
as with a, ß,and 7 isotype-specific PKC antibodies, showed
that MCF-7/ADR cells contained highly elevated amounts of
PKC«when compared to that of MCF-7/WT cells, while no
difference was observed in the amounts of PKC/i, and PKC7
was not detected in these cell lines; and (/) immunoblot analysis
of proteins extracted from purified nuclei isolated from these
MCF-7 cell lines revealed that nuclei isolated from MCF-7/
ADR cells contained highly elevated levels of PKCa.
Protein kinase C has been found to be associated with the
nuclear fraction of a number of cell types (18, 27-32). To
establish that the markedly elevated level of PKC found in
MCF-7/ADR cells represents a pool of PKC separate from the
cytosolic and plasma membrane fractions of PKC, we carried
out time course studies of changes in the distribution and downregulation of PKC activity in the different subcellular fractions
induced by treatment of these cells with 1 ^M TPA (Fig. 3).
When PKC activity had been down-regulated by prolonged (24h) exposure of MCF-7/ADR cells to TPA, significant PKC
activity still remained in both the cytosolic (20% of control)
and plasma membrane (40% of control) subcellular fractions.
In contrast, there was a complete loss of the nuclear pool of
PKC activity within 12 h after exposure of the cells to TPA.
This, along with the differences noted in the rapid (30-min)
redistribution of PKC from the cytosolic to the plasma mem
brane fraction, compared with no change detected in the nuclear
PKC activity at 30 and 90 min of TPA treatment, strongly
suggests that the nuclear pool of PKC present in MCF-7/ADR
cells is distinct from the cytosolic and plasma membrane pools
of PKC.
There appears to be a good correlation between the level of
nuclear PKC activity found and the degree of drug resistance
in MCF-7 human breast carcinoma cells. This was observed in
two different MCF-7 cell lines: (a) MCF-7 cells obtained from
Dr. Kenneth Cowan [control MCF-7/WT cells and multidrugresistant MCF-7/ADR cells (MCF-7/ADR'1 and MCF-7/
ADRjm)]; (b) MCF-7 cells obtained from Dr. Grace Yeh [control
MCF-7(GY)/WT cells and multidrug-resistant MCF-7(GY)/
W0.3 cells and MCF-7(GY)/W10 cells]. In both MCF-7 cell
lines the most resistant cell types were found to contain the
highest level of nuclear PKC activity, with less resistant cell
types having intermediate levels of nuclear PKC activity relative
to the very low level of nuclear PKC activity found in the
control, drug-sensitive cell types. It should be noted that this
quantitative correlation between the level of nuclear PKC and
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ELEVATED NUCLEAR PKC AND DRUG RESISTANCE
the degree of drug resistance is masked when PKC activities
determined in total cell homogenates are compared. A close
relationship between the level of PKC activity present in the
cytosol and plasma membrane and the degree of drug resistance
was not evident.
Observations made during the course of this investigation
indicate that the PKC activities found in the purified nuclear
fractions isolated from MCF-7/WT and MCF-7/ADR cells
exhibit somewhat different characteristics, suggesting that a
different or modified form of PKC may be present in the nucleus
of MCF-7/ADR cells, (a) The DEAE-cellulose column elution
profiles of nuclear PKC isolated from MCF-7/WT and MCF7/ADR cells revealed both quantitative and qualitative differ
ences between these two activities (Fig. 5). With MCF-7/WT
cells, the low amount of nuclear PKC activity detected was
eluted with 140 ITIMNaCl. In contrast, two major peaks of
nuclear PKC activity were observed with the extract from MCF7/ADR cells. The first peak eluted at 140 HIMNaCl, while the
second, more prominent, peak of MCF-7/ADR nuclear PKC
activity eluted at 180 HIMNaCl. The explanation for the altered
elution profile for the second, high salt peak of PKC activity
noted with the nuclear protein fraction isolated from MCF-7/
ADR cells remains to be established. Both changes in the
phosphorylation state of PKC (33) and changes in the oxidative
state of PKC (34, 35) have been reported to modify PKC to a
form which élûtes
from DEAE-cellulose at higher (200-250
HIM) salt concentrations. Modifications of this type may be
responsible for the altered form of PKC found in nuclei isolated
from MCF-7/ADR cells, and these possibilities are currently
under investigation, (b) When the CHAPS-solubilized nuclear
protein fractions of MCF-7/WT and MCF-7/ADR cells were
electrophoresed using 10-20% polyacrylamide gradient gels,
Western blot analysis revealed a distinct difference in both the
intensity and pattern of PKC immunostaining between the two
cell types (Fig. 8). The upper, highly stained PKC band found
in the nuclear extract of MCF-7/ADR cells was not detectable
in the nuclear fraction of MCF-7/WT cells, (c) Our preliminary
data indicate that the nuclear pool of PKC present in MCF-7/
ADR cells is rapidly inactivated in response to treatment of
these multidrug-resistant cells with H2O2 and with sangivamycin. Neither of these treatments results in a loss of PKC activity
from the nuclear fraction of drug-sensitive MCF-7/WT cells,
further indicating a difference in these nuclear PKC pools.4
In conclusion, the results presented indicate that drug-resist
ant MCF-7/ADR cells contain not only increased amounts of
cytosolic PKC but also markedly elevated levels of an appar
ently modified form of PKCa at the nucleus. However, it
remains to be determined what role this increase in nuclear
PKC might play in the development and modulation of multidrug resistance. Nuclear proteins such as histone H1 (36), DNA
methyltransferase (37), topoisomerase II (38), poly(ADP-ribose) synthetase (39), DNA polymerase a (40), and lamin B
(41) all have been tentatively identified as possible substrates
for phosphorylation by PKC. In addition, studies have been
reported which support a role for PKC in the regulation of
selected gene expression (42-44). Thus, it is conceivable that
the elevated cytosolic PKC noted in drug-resistant MCF-7/
ADR cells may catalyze the phosphorylation of proteins such
as the gpl70 transporter protein (25, 26) to mediate drug
resistance, while the nuclear pool of PKC found in MCF-7/
ADR cells may play a role in altering the expression of gene(s)
' Manuscripts in preparation.
important for either the onset or the maintenance of the drugresistant state of these cells.
ACKNOWLEDGMENTS
We are grateful to Dr. Kenneth Cowan (National Cancer Institute,
NIH, Bethesda, MD) for generously providing us MCF-7/WT cells and
MCF-7/ADR cells, and to Dr. Grace Yeh (National Cancer Institute)
for kindly providing us MCF-7(GY)/WT cells, MCF-7(GY)/W0.3, and
MCF-7(GY)/W 10 cells. We also wish to thank Dr. Ulf Rapp (National
Cancer Institute, Frederick County Research Facility, NIH, Frederick,
MD) and Dr. Peter Blumberg (National Cancer Institute) for help and
advice in the preparation and characterization of the PKC antisera used
in this study.
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3759
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Elevated Level of Nuclear Protein Kinase C in
Multidrug-resistant MCF-7 Human Breast Carcinoma Cells
Sung Ae Lee, James W. Karaszkiewicz and Wayne B. Anderson
Cancer Res 1992;52:3750-3759.
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