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J. Biol. Chem., Vol. 277, Issue 35, 31407-31415, August 30, 2002
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Reduces
Topoisomerase II Catalytic Activity, Cleavable Complexes Formation, and
Drug-induced Cytotoxicity in Monocytic U937 Leukemia Cells*
§,,,
**
From the INSERM E9910, Institut Claudius
Régaud, 20 rue du Pont Saint Pierre, 31052 Toulouse cedex,
France, the ¶ Laboratory of Molecular Pharmacology, NCI,
National Institutes of Health, Bethesda, Maryland
20892-4255, and the Service d'Hématologie,
Centre Hospitalier Universitaire Purpan, 31059 Toulouse, France
Received for publication, May 13, 2002, and in revised form, June 19, 2002
 |
ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
In this study, we evaluated the influence of
protein kinase C DNA topoisomerases II are nuclear enzymes that modify DNA topology
by their ability to break and reseal both strands in concert. Topoisomerases II have important functions in DNA replication and can
serve as a cancer chemotherapy target. Indeed, drugs such as etoposide
(VP-16) or mitoxantrone, form drug-topoisomerase II-DNA ternary
complexes referred to as "cleavable complex." The primary
cytotoxic effect of these so-called "topoisomerase II inhibitors"
is not by inhibition of topoisomerase II activity but rather by
stabilizing topoisomerase II cleavable complexes. This interaction
prevents the DNA-resealing step normally catalyzed by topoisomerase II.
The ternary complex constitutes a latent DNA-damaging state, which is
ultimately converted to an irreversible DNA double-strand break
(DSB).1 Although the
mechanism by which complex formation mediates cell death is still
poorly understood, it has been largely documented with few exceptions
that the amount of cleavable complexes and the subsequent number of DNA
breaks correlates with cytotoxicity (1). These observations suggest
that abnormal intracellular distribution or a decrease in expression
level, activity, and sensitivity of the inhibited topoisomerase may
have major impacts on topoisomerase inhibitor clinical efficacy. This
has been confirmed by the molecular characterization of the
so-called atypical multidrug resistant phenotype (at-MDR) resulting
from selection by topoisomerase II inhibitors. Indeed, at-MDR cells
display cross-resistance to other topoisomerase II inhibitors and have
been associated with a number of functional and/or structural
topoisomerase II alterations, including decreased catalytic
activity, abnormal interaction between topoisomerase II and nuclear
matrix, reduced expression, point mutation and, finally, altered
phosphorylation (2).
The role of phosphorylation on topoisomerase II function has been
debated and remains controversial. Indeed, previous studies have shown
that topoisomerase II contains potential serine phosphorylation sites
and that this enzyme is a substrate for various serine kinases, including casein kinase II, p34cdc2 kinase, and classic
protein kinase C (PKC). In a cell-free system, PKC-induced
phosphorylation of topoisomerase II results in an increase in its
catalytic activity by enhancing ATP hydrolysis (3, 4). In the absence
of antineoplastic drugs, phosphorylation has a negligible effect on
other steps of topoisomerase II catalytic cycle, including DNA binding
or DNA cleavage/religation equilibrium. However, in the presence of
VP-16 or amsacrine, phosphorylation decreases the ability of drugs to
stabilize DNA-topoisomerase II complexes, apparently by increasing the
rates of religation of DNA by the enzyme (5). Other studies have
provided indirect evidences that PKC might also influence topoisomerase
II function in vivo. For example, PKC inhibitors, such as
suramin or staurosporine, decrease topoisomerase II phosphorylation and
catalytic activity in intact cells as well as drug-induced
topoisomerase II-mediated cleavage (6, 7). However, the role of
topoisomerase II phosphorylation in drug resistance has been minimized
on the basis of independent studies that have shown that, in at-MDR
cells, topoisomerase II could be either hyperphosphorylated or
hypophosphorylated (8-10).
At least 12 different isoforms of PKC have been characterized so far
and have been separated into three categories based on the
Ca2+ requirement for activation and phorbol ester binding
activity. Conventional PKCs ( PKC Materials--
Recombinant PKC Cell Culture--
U937 cells were transfected by electroporation
at 0.25 kV and 960 farads either with 20 µg of the PKC MTT Assay--
This assay is based on the ability of viable
mitochondria to convert MTT, a soluble tetrazolium salt
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) into an
insoluble formazan precipitate, which is dissolved in dimethyl
sulfoxide and quantified by spectrophotometry. Cells (30,000) were
seeded in 96-well plates and treated with cytotoxic agents for 48 h. Absorbance corresponding to MTT conversion was read at two
wavelengths, 540 and 690 nm.
DNA Filter Elution Assays--
Exponential growing cells were
labeled with [3H]thymidine (0.02 µCi/ml) for 48 h,
chased for 2 h in isotope-free medium, and exposed to VP-16 for
the indicated time. Equal numbers of cells (5 × 105)
were loaded onto polycarbonate or PVC filters, lysed, and subjected to
elution (20). Radioactivity in the DNA fractions was counted, and the
fraction of the DNA retained on the filter was calculated as follows:
fraction retained/(filter plus lysis plus fraction retained). Elution
of DNA through polycarbonate filters reflects the presence of DSB, and
elution of DNA through PVC filters reflects the presence of DNA-protein
cross-link (DPC). DPC frequency (in rad-equivalents) was computed
according to the formula: DPC = [(1 Protein Analysis--
For cytoplasmic protein, 1 × 107 cells were washed twice in phosphate-buffered saline
and lysed by resuspension in lysis buffer containing 10 mM
HEPES (pH 7.8), 100 mM EDTA, 100 mM EGTA, 1 mM PMSF, 2 µM pepstatin A, 0.6 µg/ml
aprotinin on ice for 10 min. Nonidet P-40 (0.3%) was then added for 2 min, and the cytoplasmic lysate (supernatant) was collected after
centrifugation at 10,000 × g for 2 min at 4 °C. For
nuclear lysate, 1 × 107 cells were washed twice in
phosphate-buffered saline and lysed by resuspension in lysis buffer
containing 10 mM HEPES (pH 7.8), 100 mM EDTA,
100 mM EGTA, 1 mM PMSF, 2 µM
pepstatin A, 0.6 µg/ml aprotinin on ice for 10 min. Nonidet P-40 was
then added at 0.3% final for 5 min, and the nuclear pellet was
resuspended in 20 mM HEPES (pH 7.8), 400 mM
NaCl, 1 mM EDTA, 1 mM EGTA. Aliquots were
sonicated and centrifuged at 20,000 × g for 10 min at
4 °C, and supernatants containing nuclear proteins were collected.
Nuclear or total cell lysates were resuspended in a denaturing loading buffer, and proteins were loaded in SDS-PAGE (7.5 or 10%), transferred onto nitrocellulose, and probed with anti-topoisomerase II Preparation of Nuclear Extracts--
Cells (1 × 107) were washed once with phosphate-buffered saline and
twice with buffer A (1 mM KH2PO4, 5 mM MgCl2, 150 mM NaCl, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 2 µg/ml leupeptin, 2 µg/ml aprotinin) and resuspended for 10 min with
buffer C containing 0.3% Triton X-100. Cells were then centrifuged and
resuspended in 100 µl of buffer A. One-hundred microliters of buffer
A containing 0.55 M NaCl was then added. After mixing and
gentle rotation for 30 min at 4 °C, samples were centrifuged for 10 min at 14,000 rpm. Supernatants were used as nuclear extracts.
Topoisomerase II Decatenation Assay--
Decatenation assays
were carried out by incubating 0.25 µg of kinetoplast DNA with
nuclear extracts or recombinant proteins in buffer B containing 10 mM Tris-HCl, pH 7.9, 50 mM NaCl, 50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 15 µg/ml bovine serum albumin, 1 mM
ATP. After 30 min at 30 °C, reactions were quenched by addition of
1% SDS, 0.5% bromphenol blue, and 30% glycerol. DNA products were
resolved on 1% agarose-Tris borate-EDTA gels at 16 V overnight. Agarose gels were stained with ethidium bromide, and fluorescence was
quantified by UV imager.
Topoisomerase II Phosphorylation Analysis--
Extracts were
prepared by lysing cells (15 × 106) in buffer C
containing 50 mM HEPES, pH 7.0, 1 mM EDTA, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 10 mM
Na4P2O7, 100 mM NaF, 1 mM NaVO3, 10 µg/ml leupeptin, 10 µg/ml
aprotinin, 1 mM PMSF, and 10 mM DTT. Cells
extracts (1.5 mg) were sonicated, clarified, and immunoprecipitated
with 3 µg of anti-phosphoserine antibody overnight at 4 °C. Immune
complexes were collected by incubation with protein G-Sepharose beads
for 60 min at 4 °C. The beads were extensively washed with buffer D
containing 50 mM HEPES, pH 7.0, 10% glycerol, 0.1% Triton
X-100, 1 mM NaVO3, 150 mM NaCl).
Denaturing loading buffer was added to immune complexes and boiled for
5 min, and the samples were loaded for SDS-PAGE analysis (7.5%), transferred onto a nitrocellulose membrane, and probed with
anti-topoisomerase antibodies. Proteins were detected by chemiluminescence.
Co-immunoprecipitation of PKC PKC Phosphorylation of Topoisomerase II Influence of PKC Influence of PKC Influence of PKC Influence of PKC Influence of PKC Interaction between PKC Influence of PKC Interaction between rH-PKC Influence of rH-PKC This study shows that PKC Based on the kinase function of PKC With regard to topoisomerase II The role of atypical PKC isoforms, including PKC To conclude, we propose a model in which, upon PKC
(PKC) on topoisomerase II inhibitor-induced
cytotoxicity in monocytic U937 cells. In U937-J and U937-B cells,
enforced PKC expression, conferred by stable transfection of PKC
cDNA, resulted in total inhibition of VP-16- and
mitoxantrone-induced apoptosis and decreased drug-induced cytotoxicity,
compared with U937-neo control cells. In PKC-overexpressing cells,
drug resistance correlated with decreased VP-16-induced DNA strand
breaks and DNA protein cross-links measured by alkaline elution.
Kinetoplast decatenation assay revealed that PKC overexpression
resulted in reduced global topoisomerase II activity. Moreover, in
PKC-overexpressing cells, we found that PKC interacted with both
and 
isoforms of topoisomerase II, and these two enzymes were
constitutively phosphorylated. However, when human recombinant PKC
(rH-PKC) was incubated with purified topoisomerase II isoforms,
rH-PKC interacted with topoisomerase II but not with
topoisomerase II. PKC/topoisomerase II interaction resulted in
phosphorylation of this enzyme and in decrease of its catalytic
activity. Finally, this report shows for the first time that
topoisomerase II is a substrate for PKC, and that PKC may
significantly influence topoisomerase II inhibitor-induced cytotoxicity
by altering topoisomerase II activity through its kinase function.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, I, II, and 
) are
Ca2+-dependent phorbol ester receptor kinases;
novel PKCs (
, 
, 
, and 
) are Ca2+-independent
phorbol ester receptor kinases; and atypical PKCs (, 
, 
, and
) are independent of both Ca2+ and phorbol ester.
Previous studies have shown that topoisomerase II is phosphorylated
in vitro by each of the conventional PKC isoforms (11).
However, the influence of these PKC isozymes on cellular topoisomerase
function in vivo is still largely unknown. Moreover, to the
best of our knowledge, the influence of atypical PKC isozymes on
topoisomerase II phosphorylation and function has not been investigated.
is an atypical PKC isoform, which is activated directly
or indirectly by a variety of important signaling molecules, including
ceramide (12, 13), phosphatidic acid (14), and diacylglycerol generated
from phosphatidylcholine hydrolysis (15), phosphoinositide 3-kinase
lipid products (16), and p21Ras (17). PKC has emerged as a critical
regulator of a number of cellular functions, including proliferation,
differentiation, and apoptosis inhibition (18). Despite the critical
role of this enzyme in cellular signaling, its implication in the
regulation of topoisomerase II function has never been examined. This
study was aimed to evaluate the effect of PKC overexpression on the
formation of cleavable complexes and cytotoxicity induced by VP-16 in
the human leukemic U937 cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and purified PKC containing
, , and isoforms were purchased from Calbiochem (San Diego,
CA). Myelin basic protein (MBP) was from Sigma (St. Quentin-Fallavier,
France). Anti-topoisomerase II and anti-topoisomerase II antibodies were obtained from Santa-Cruz/TEBU (Le Perray en Yvelines,
France). Anti-PKC antibody was purchased from UBI/Euromedex
(Souffelweyersheim, France). Anti-phosphoserine was obtained from
Zymed Laboratories Inc. (Montrouge, France).
Topoisomerase II was from USB (Cleveland, OH), and topoisomerase II and were from Dr. Y. Pommier (NCI, National Institutes of
Health, Bethesda, MD). Kinetoplast DNA was from TopoGen (Columbus, OH).
[-32P]ATP (7000 Ci/mmol) was purchased from ICN
(Orsay, France). [methyl-3H]Thymidine (79 Ci/mmol) and an ECL detection system were from Amersham Biosciences
(Les Ullis, France).
plasmid
(corresponding to full-length rat PKC) or 20 µg of the vector
without the PKC insert using a Bio-Rad Gene Pulser as previously
described (19). For this study, two clones, U937-J and U937-B,
were selected and were compared with control U937-neo cells. Cells were
cultured in RPMI complemented with 10% fetal calf serum. U937-J,
U937-B, and U937-neo cells, displayed similar growth kinetics with a
doubling time of about 25 h. PKC overexpression resulted in a
2.5-fold increase in PKC activity as measured by MBP phosphorylation
after immunoprecipitation with anti-PKC antibody.
retentiontreated
cells)
1 (1 retentioncontrol
cells)1] × 3000.
and/or or anti-PKC antibodies. Immune complexes were detected by using the chemiluminescent detection system.
and Topoisomerase
II--
Extracts were prepared by lysing cells (15 × 106) in buffer C. Cells extracts (1.5 mg) were sonicated,
clarified, and immunoprecipitated with 3 µg of anti-PKC antibody
overnight at 4 °C. Immune complexes were collected by incubation
with protein G-Sepharose beads for 60 min at 4 °C. The beads were
then extensively washed with buffer D. For in vitro
interaction, recombinant PKC was preincubated with
topoisomerase II for 1 h at 32 °C in buffer B and
immunoprecipitated with 3 µg of anti-PKC antibody overnight at
4 °C. Immune complexes were collected with protein A-Sepharose beads
for 60 min at 4 °C. The beads were then washed with buffer B. Denaturing loading buffer was added to immune complexes from in
vivo or in vitro experiments. Samples were boiled for 5 min, run in SDS-PAGE (7.5%), transferred onto nitrocellulose membrane,
and probed with anti-topoisomerase II antibodies. Proteins were
detected by chemiluminescence.
Activity--
Cells (5 × 106) were
lysed in 20 mM HEPES, pH 7.4, 0.5 M EDTA, 125 mM NaCl, 0.1% Nonidet P-40, 1 mM
NaVO3, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM PMSF, 0.5 mg/ml benzamidine, and 1 mM DTT.
Cell extracts (300 µg) were immunoprecipitated with 3 µg of
anti-PKC antibody overnight at 4 °C. Immune complexes were collected by incubation with protein G-Sepharose beads for 60 min at
4 °C. The beads were extensively washed four times with lysis
buffer, twice with kinase buffer (20 mM HEPES, pH 7.4, 1 mM DTT, 25 mM NaCl) and finally incubated with
a buffer containing 20 mM HEPES, pH 7.4, 10 mM
MgCl2, 1 mM DTT. For in vitro
experiments, recombinant proteins were incubated in buffer B. Kinase
assays were performed for 15 min at 32 °C using MBP as substrate and 10 µCi of [-32P]ATP. Reactions were stopped by
addition of 15 µl of 2× SDS buffer and boiled for 5 min, and the
samples were loaded for SDS-PAGE analysis (10%). Phosphorylated MBP
levels were analyzed by autoradiography.
by PKC
--
Recombinant PKC was preincubated with topoisomerase II for 1 h at 32 °C in 20 µl of buffer B and 10 µCi of
[-32P]ATP and 4 µg of phosphatidylserine. Reactions
were stopped by addition of 5 µl of 4× SDS buffer and boiled for 5 min, and the samples were loaded for SDS-PAGE (7.5%). Phosphorylated
topoisomerase II levels were analyzed by autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Overexpression on Topoisomerase II
Inhibitor-induced Cytotoxicity in U937 Cells--
U937-neo cells and
two clones overexpressing PKC, U937-J, and U937-B, were
treated with the topoisomerase II inhibitors VP-16 and mitoxantrone,
and cell viability was evaluated 48 h later using the MTT assay.
U937-J or U937-B cells were 5-fold more resistant than U937-neo
cells to VP-16 (Fig. 1A).
PKC overexpression conferred an even more efficient protection
against mitoxantrone-induced cytotoxicity (Fig. 1B). PKC
overexpression was also found to inhibit VP-16- and
mitoxantrone-induced apoptosis as evaluated by
4',6-diamidino-2-phenyl-indole staining (Fig. 1, C and
D). These results showed that PKC overexpression
conferred resistance to topoisomerase II inhibitors. We then
investigated the influence of PKC overexpression on
VP-16-induced DNA damage.
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Fig. 1.
Influence of PKC
overexpression on topoisomerase II inhibitor-induced cytotoxicity
in U937 cells. U937-neo cells 
, U937-J cells (
), and
U937-B cells (
) were treated either with VP-16 (A,
C) or mitoxantrone (B, D) for 48 h. Cell viability was assayed by MTT assay (A,
B), and apoptotic cells were determined by
4',6-diamidino-2-phenyl-indole staining (C, D).
Results are means ± S.D. of three independent experiments.
Overexpression on VP-16-induced DNA Strand
Breaks in U937 Cells--
U937-neo and U937-J cells were prelabeled
with [3H]thymidine for 48 h, chased with fresh
medium, and treated with VP-16 for 1 h and DSB were determined
using alkaline elution (20, 21). As shown in Fig.
2, VP-16 produced significantly less DNA
DSB in U937-J cells than in U937-neo cells. VP-16-induced DPC were also compared in U937-neo and U937-J cells. As shown in Fig. 3, whereas VP-16 induced DPC in a
dose-dependent manner in both U937-neo and U937-J cells,
the levels of DPC were significantly lower in U937-J. These results
showed that PKC overexpression resulted in reduced VP-16-induced DNA
damage. To rule out the possible influence of PKC on drug transport,
we measured VP-16-induced DPC in isolated nuclei from U937-neo and
U937-J cells. As shown in Fig. 4,
VP-16 produced a dose-dependent increase in DPC in U937-neo nuclei,
whereas there was no detectable DPC formation in U937-J nuclei. This
result confirmed that PKC overexpression resulted in a significant
reduction in VP-16-induced DNA damage, which could not be explained by
altered drug transport.
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Fig. 2.
Influence of PKC
overexpression on VP-16-induced DNA DSB in U937 cells.
U937-neo cells (
, , 
) and U937-J cells (
, , 
) were
untreated (, ) or treated with VP-16 for 1 h at 50 µM (, ) or 100 µM (, ). DNA
retention rate was determined using alkaline elution short method (20,
21) as described under "Experimental Procedures."
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Fig. 3.
Influence of PKC
overexpression on VP-16-induced DPC in U937 cells. U937-neo
cells () or U937-J cells () were treated with various doses of
VP-16 for 1 h. DPC were calculated using the long alkaline elution
method as described under "Experimental Procedures." Results are
means ± S.D. of three independent experiments. Statistical
analyses were performed using the Student's t test, and the
asterisk represents significant differences
(p < 0.05) compared with U937-neo cells.
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Fig. 4.
Influence of PKC
overexpression on VP-16-induced DPC in U937-isolated nuclei.
Isolated U937-neo () or U937-J () nuclei were treated with
various doses of VP-16 for 1 h. DPC were calculated using alkaline
elution as described under "Experimental Procedures." Results are
means ± S.D. of three independent experiments. Statistical
analyses were performed using the Student's t test, and the
asterisk represents significant differences
(p < 0.05) compared with U937-neo cells.
Overexpression on Topoisomerase II Expression
in U937 Cells--
To investigate the possible influence of PKC on
topoisomerase II expression, U937-neo, U937-J, and U937-B cell
extracts were analyzed by Western blotting with anti-topoisomerase
II and anti-topoisomerase II antibodies. As shown in Fig.
5, topoisomerase II and topoisomerase
II expression levels in U937-neo, U937-J, and U937-B cells
were comparable. This result suggests that reduced VP-16-induced DNA
damage in PKC-overexpressing cells was not due to decreased
topoisomerase II expression. For this reason, we hypothesized that
PKC overexpression may result in reduced topoisomerase II
activity.
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Fig. 5.
Influence of PKC
overexpression on topoisomerase II expression in U937 cells.
Whole U937-neo, U937-J, and U937-B cell extracts were subjected
to immunoblot analysis with anti-topoisomerase II and
anti-topoisomerase II antibodies. Data are from one
experiment representative of three experiments.
Overexpression on Topoisomerase II Activity in
U937 Cells--
Decatenation of kinetoplast DNA was used as a specific
assay to evaluate topoisomerase II activity of nuclear extracts
prepared from U937-neo, U937-J, and U937-B cells (22). Nuclear
extracts from U937-neo cells exhibited topoisomerase II activity in a
dose range comprised between 500 and 1000 ng of total nuclear protein, whereas topoisomerase II activities contained in U937-J and
U937-B preparations were dramatically reduced (Fig.
6A). Based on Western blot
analysis of nuclear cell extracts, it appeared that nuclear PKC
expression was inversely correlated with topoisomerase II activity
(Fig. 6B). For this reason, we hypothesized that PKC inhibited topoisomerase II activity by influencing topoisomerase II
phosphorylation.
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Fig. 6.
Influence of PKC
overexpression on topoisomerase II activity in U937 cells.
A, topoisomerase II activity of nuclear extracts (500 or
1000 ng of proteins) from U937-neo, U937-J, and U937-B cells was
measured by decatenation of kinetoplast DNA as a specific assay for
topoisomerase II activity. B, nuclear extracts from
U937-neo, U937-J, and U937-B cell extracts were subjected to
immunoblot analysis with anti-PKC. Data are from one experiment
representative of three experiments.
Overexpression on Serine Phosphorylation of
Topoisomerase II in U937 Cells--
U937-neo, U937-J, and U937-B
cell extracts were immunoprecipitated with anti-phosphoserine antibody,
and topoisomerase II was immunoblotted with anti-topoisomerase II
and anti-topoisomerase II antibodies. As shown in Fig.
7, U937-J and U937-B cells exhibited constitutive topoisomerase II and serine
hyperphosphorylation, compared with U937-neo cells, whereas
anti-phosphothreonine antibody, used as control, was not reactive. This
result shows that PKC increased phosphorylation of both isoforms of
topoisomerase II.
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Fig. 7.
Influence of PKC
overexpression on serine-phosphorylation of topoisomerase II in
U937 cells. U937-neo, U937-J, and U937-B cell extracts were
immunoprecipitated with anti-phosphoserine antibody and topoisomerase
II was immunoblotted with anti-topoisomerase II and
anti-topoisomerase II antibodies. Data are from one experiment
representative of three experiments.
and Topoisomerase II in U937-neo and
PKC Overexpressing U937 Cells--
PKC/topoisomerase II
interaction was assessed by immunoprecipitation. However, because
anti-topoisomerase II- and -specific antibodies used in this
study were not suitable for immunoprecipitation, cellular extracts of
U937-neo, U937-J, and U937-B were immunoprecipitated with
anti-PKC antibody and topoisomerase II and proteins were immunoblotted with relevant antibodies. In U937-neo cell extracts, we
found that PKC interacted neither with topoisomerase II nor with
topoisomerase II, although a significant PKC amount was detected
in the immunoprecipitates (Fig. 8).
However, in U937-J and U937-B cellular extracts, both
topoisomerase II and co-immunoprecipitated with PKC (Fig. 8).
These results suggest that, in PKC-overexpressing U937 cells, the
enzyme may directly or indirectly interact with both topoisomerase
II and isoforms and that these interactions seriously interfere
with topoisomerase II activity. To investigate this hypothesis, we
evaluated in a cell-free system the influence of recombinant PKC on
the activity of purified topoisomerase II preparations containing both
topoisomerase II and .
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Fig. 8.
Interaction between PKC
and topoisomerase II in U937 cells. U937-neo, U937-J, and
U937-B cell extracts were immunoprecipitated with anti-PKC
antibody and topoisomerase II (A) or PKC (B)
were immunoblotted with anti-topoisomerase II and anti-topoisomerase
II antibodies or with anti-PKC antibody, respectively. Data are
from one experiment representative of three experiments.
on Purified Topoisomerase II Activity in a
Cell-free System--
In these experiments, recombinant human PKC
(rH-PKC) (1 or 3 µg) was incubated with topoisomerase II (50 ng),
and topoisomerase II activity was measured by the decatenation assay.
As shown in Fig. 9A, PKC
was found to inhibit topoisomerase II activity in a
dose-dependent manner. In contrast, a mixture containing
PKC, PKC, and PKC was found to stimulate topoisomerase II
activity as previously described (Fig. 9B) (11). This
result suggests that PKC does interfere with either topoisomerase
II or activities. To further investigate this finding, we
evaluate the capacity of PKC to interact with purified topoisomerase
II or .
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Fig. 9.
Influence of rH-PKC
on purified topoisomerase II activity in a cell-free system.
rH-PKC (1 or 3 µg) (A) or PKC, , (50 ng)
(B) were incubated with topoisomerase II (50 ng) for 1 h at 32 °C, and topoisomerase II activity was measured by the
decatenation of the kinetoplast DNA as a specific assay for
topoisomerase II activity. Data are from one experiment representative
of three experiments.
and Purified
Topoisomerase II Isoforms in a Cell-free System--
rH-PKC was
co-incubated with either topoisomerase II or topoisomerase II in
a molar ratio of 2:1. PKC/topoisomerase II complexes were
immunoprecipitated using anti-PKC antibody, and topoisomerase
isoforms were revealed by immunoblotting with specific anti-topoisomerase antibodies. Controls were provided by
immunoblotting immunoextracts with anti-PKC (Fig.
10A). As shown in Fig.
10B, a small amount of topoisomerase II nonspecifically
bound to protein A-Sepharose. However, the level of topoisomerase II
detected following immunoprecipitation with anti-PKC antibody was
significantly increased, suggesting that most of topoisomerase II
specifically bound to PKC. In contrast, despite many efforts, we
were unable to detect topoisomerase II in anti-PKC immunoextracts
(data not shown). These results showed that PKC was able to interact with topoisomerase II but not topoisomerase II. The consequence of this interaction on topoisomerase II phosphorylation was also investigated. Topoisomerase II (1 µg) was incubated with rH-PKC (1 µg) in the presence of [-32P]ATP and
phosphatidylserine, and phosphorylated topoisomerase II was
revealed by autoradiography. PKC kinase activity was checked using
MBP as a substrate (Fig. 10C). Topoisomerase II was
found to be constitutively phosphorylated. However, incubation with
PKC resulted in a 2-fold increase in topoisomerase II
phosphorylation (Fig. 10D). Altogether, these results
demonstrated that PKC did interact with topoisomerase II and
phosphorylated this enzyme.
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Fig. 10.
Interaction between
rH-PKC and purified topoisomerase
II isoform in a cell-free system.
rH-PKC (1 µg) was incubated with topoisomerase II (1 µg) for
1 h at 32 °C and PKC/topoisomerase II complexes were
subjected to immunoprecipitation using anti-PKC antibody following
by immunoblotting with either anti-PKC antibody (A) or
anti-topoisomerase II (B). PKC kinase activity was
checked using MBP as a substrate as described under "Experimental
Procedures" (C). Topoisomerase II (1 µg) was
incubated with rH-PKC (1 µg) for 1 h at 32 °C in the
presence of [-32P]ATP and phosphatidylserine (4 µg),
and phosphorylated topoisomerase II was revealed by autoradiography
after separation in a SDS-PAGE (7.5%) (D). Data are from
one experiment representative of three experiments.
on Topoisomerase II
Activity--
In these experiments, rH-PKC was incubated with
topoisomerase II at a molar ratio of 2:1, and topoisomerase II
activity was measured by the decatenation assay. As shown in Fig.
11, PKC was found to inhibit
topoisomerase II activity.
View larger version (48K):
[in a new window]
Fig. 11.
Influence of rH-PKC
on purified topoisomerase II
activity. rH-PKC (530 ng) was incubated with
topoisomerase II (650 ng) for 1 h at 32 °C, and
topoisomerase II activity was measured by the decatenation assay.
Data are from one experiment representative of three experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
overexpression in U937
cells resulted in inhibition of apoptosis and increased survival of
U937 cells treated with VP-16 and mitoxantrone, two topoisomerase
II inhibitors. Enforced PKC expression resulted in a marked decrease in VP-16-induced DPC and DNA DSB, whereas the level of topoisomerase II and topoisomerase II expression was unchanged compared with control cells. These results suggest that PKC can interfere with topoisomerase II function. In fact, we found that
PKC-overexpressing cells exhibited reduced topoisomerase II
catalytic function as measured by the decatenation assay. Altered
topoisomerase II catalytic cycle may explain reduced drug-induced DNA
damage and cytotoxicity. Thus, this study shows for the first time that
a specific PKC isozyme may inhibit topoisomerase II catalytic activity
and VP-16-induced apoptosis and cytotoxicity by interfering with
drug-induced DNA damage.
, we hypothesized that PKC
overexpression might result in abnormal topoisomerase II
phosphorylation. In fact, we found that, in PKC-overexpressing
cells, PKC was not only found to interact with topoisomerase
II and topoisomerase II but also that these two topoisomerase II
isoforms were heavily phosphorylated on serine residues. These results
suggest that, in PKC-overexpressing cells, PKC not only directly
or indirectly interacts with the two topoisomerase II isoforms but also
phosphorylates these enzymes. However, using a cell-free system, we
described that only topoisomerase II is a substrate for PKC and
that PKC inhibits topoisomerase II activity. This result suggests
that, in PKC-overexpressing cells, PKC interacts directly with
topoisomerase II and inhibits topoisomerase II catalytic
activity. This hypothesis is consistent with the role of this
topoisomerase II form in the cytotoxicity of topoisomerase II
inhibitors (23, 24).
, the fact that this enzyme was found
to interact in vivo, but not in vitro, with
PKC, suggests that, in PKC-overexpressing cells,
PKC/topoisomerase II interaction involves one or several other
proteins required for the constitution of this complex. Moreover, the
fact that, in PKC-overexpressing cells, topoisomerase II was
found to be constitutively phosphorylated whereas, in vitro,
PKC was unable to phosphorylate this enzyme, suggests that
topoisomerase II is phosphorylated by another PKC-regulated kinase. In this perspective, it is interesting to note that in a recent
study topoisomerase II was found to be phosphorylated in intact
cells by ERK2, the effector serine kinase of the classic MAPK module
(25). Based on previous studies, which have documented that PKC is a
downstream target of MAPK (26, 27), topoisomerase II phosphorylation
could result from PKC-mediated ERK2 activation in
PKC-overexpressing cells. The fact that, in these cells, ERK2 was
found to be constitutively activated and accumulated in the nucleus
(data not shown) supports this hypothesis.
, in cell survival
has been previously documented. Indeed, it has been described that the
blockade of PKC or PKC/ with dominant-negative mutants or
antisense oligonucleotides is sufficient to promote apoptosis (28, 29).
The inactivation of PKC by caspase-dependent proteolysis during apoptosis induced by UV (30) or by cisplatin (31) strengthens the role of PKC in the cellular protection against genotoxic stress.
The mechanism by which atypical PKC isoforms exert their anti-apoptotic
effect has received a great deal of attention. These studies strongly
suggested that NF-
B signaling pathways could play an important role
in PKC-induced inhibition of apoptosis (32). Indeed, NF-B is a
negative regulator of apoptosis induced by genotoxic agents, including
topoisomerase II inhibitors (33, 34). Therefore, we cannot rule out
that PKC overexpression may result in the activation of
anti-apoptotic signals that interfere with the post-damage apoptotic
response and, therefore, contribute to drug resistance.
accumulation in
the nucleus, this enzyme interacts with and phosphorylates nuclear
topoisomerase II. Topoisomerase II hyperphosphorylation reduces catalytic function and decreases formation of ternary complexes and drug-induced cytotoxicity. If so, nuclear PKC
accumulation might function to regulate topoisomerase II function.
Although very little is known about expression and subcellular
localization of PKC in tumor cells, PKC may translocate to the
nucleus upon stimulation by differentiating agents (35), growth factors
(36, 37), cytokines (38), or hypoxia (39). Whether PKC alters topoisomerase II function in these conditions will be the subject of
further investigations.
| |
ACKNOWLEDGEMENT |
|---|
We thank Dr. Ways (Lilly
Corporate Center, USA) who kindly provided the PKC-overexpressing
U937 cell lines.
| |
FOOTNOTES |
|---|
* This work was supported in part by l'Association pour la Recherche sur le Cancer grant 5526 (to G. L.), La Faculté de Médecine Toulouse-Rangueil (to G. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ A recipient of a fellowship from la Ligue Nationale Contre le Cancer. To whom correspondence may be addressed: INSERM E9910, Institut Claudius Régaud, 20 rue du Pont Saint Pierre, 31052 Toulouse cedex, France. Tel.: 33-5-61-42-41-73; Fax: 33-5-61-42-46-06; E-mail: plo@icr.fnclcc.fr.
** To whom correspondence may be addressed: INSERM E9910, Institut Claudius Régaud, 20 rue du Pont Saint Pierre, 31052 Toulouse cedex, France. Tel.: 33-5-61-42-41-73; Fax: 33-5-61-42-46-06; E-mail: laurent@icr.fnclcc.fr.
Published, JBC Papers in Press, June 24, 2002, DOI 10.1074/jbc.M204654200
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
DSB, double-strand
breaks;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide;
at-MDR, atypical multidrug resistant phenotype;
DPC, DNA
protein cross-links;
VP-16, etoposide;
rH-PKC, recombinant human
PKC;
MBP, myelin basic protein;
PKC, protein kinase C;
PMSF, phenylmethylsulfonyl fluoride;
DTT, dithiothreitol;
MAPK, mitogen-activated protein kinase.
| |
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