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INTRODUCTION |
Leukemia is a malignant disease of the bone marrow that is
the leading cause of cancer death in children and adults under the age
of 35 (1). Leukemias arise in hematopoietic progenitor cells and are
characterized by impaired or blocked differentiation, uncontrolled
proliferation, and resistance to apoptosis (2). Many leukemias can be
effectively treated using one or combinations of chemotherapeutic
agents, such as taxol, that induce apoptosis (reviewed in Ref. 3). A
notable exception is chronic myelogenous leukemia
(CML),1 which is highly
resistant to most commonly utilized chemotherapeutic drugs, including
taxol (4, 5). CML cells, such as K562 cells, are characterized by the
presence of the Philadelphia chromosome, which results from a
reciprocal translocation involving chromosome 9 and chromosome 22 (6,
7). In K562 cells, the Abl tyrosine kinase gene on chromosome 9 is
translocated into the breakpoint cluster region (bcr) region
on chromosome 22 producing a chimeric gene whose product, Bcr-Abl,
expresses disregulated Abl tyrosine kinase activity (8). The Bcr-Abl
gene is the transforming activity responsible for CML (9), and the
resistance of K562 cells to drug-induced apoptosis is conferred by
Bcr-Abl (5, 10, 11). Thus, inhibition of Bcr-Abl tyrosine kinase
activity using selective inhibitors (12-15) or of Bcr-Abl expression
using antisense oligonucleotides (16, 17) leads to increased
sensitivity of K562 cells to apoptosis induced by many drugs including taxol.
Despite the importance of Bcr-Abl in resistance to drug-induced
apoptosis, relatively little is known about the molecular mechanisms by
which Bcr-Abl exerts its anti-apoptotic effects (18). Recently, we
demonstrated that expression of the atypical PKC isozyme, PKC
, is
important for resistance to taxol-induced apoptosis (19). We have now
further defined the role of PKC
in this process. We find that
treatment of K562 cells with taxol leads to potent and sustained
activation of PKC
. In contrast, treatment of HL60 cells, which are
sensitive to taxol-induced apoptosis, does not lead to appreciable
activation of PKC
. Furthermore, inhibition of Bcr-Abl using the
highly selective inhibitor tyrphostin AG957 blocks PKC
activation
and sensitizes K562 cells to taxol-induced apoptosis. Tyrphostin
AG957-treated cells can be rescued from taxol-induced apoptosis by
expression of constitutively active PKC
. These data, along with our
previous results (19), demonstrate that PKC
activation is both
necessary and sufficient to mediated the anti-apoptotic effects of
Bcr-Abl.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Drug Treatments--
Human K562 chronic
myelogenous leukemia cells and HL60 promyelocytic leukemia cells were
maintained in suspension culture as described previously (19). Cells at
a density of 2 × 105 cells/ml were treated with
paclitaxel (taxol) at the concentrations and for the time periods
indicated in the figure legends. In some cases, the Bcr-Abl-selective
inhibitor tyrphostin AG957 or the PI 3-kinase inhibitors wortmannin or
LY 294002 were added to K562 cell cultures at the concentrations
indicated in the figure legends prior to treatment with taxol.
Tyrphostin AG957 is a highly selective Bcr-Abl inhibitor that blocks
Bcr-Abl-mediated cell growth and resistance to apoptosis in K562 cells
(12, 20). Apoptosis was assessed by the presence of apoptotic bodies or
chromosomal condensation and by trypan blue exclusion as described
previously (19). These morphological manifestations of apoptosis
correlate directly with the apoptotic DNA fragmentation induced by
taxol and provide a convenient method to quantitate the apoptotic
response (19). For assessment of nuclear DNA morphology, 1 × 105 cells were fixed and permeablized with 1%
paraformaldehyde phosphate-buffered saline for 15 min at 37 °C,
stained with 4',6-diamidino-2-phenylindole for 5 min, and observed
under phase-fluorescence optics. A minimum of 500 cells for each
treatment were scored for the presence of apoptotic bodies.
Immunoblot Analysis for PKC
--
K562 and HL60 cell extracts
were lysed directly into SDS sample buffer and subjected to
SDS-polyacrylamide gel electrophoresis and immunoblot analysis for
PKC
as described previously (19). We have previously demonstrated
that K562 cells express PKC
but no detectable PKC
(19). Protein
quantitation was carried out using BCA protein assay reagent (Pierce).
Antigen/antibody complexes were detected using ECL (Amersham Pharmacia Biotech).
Immunoprecipitation and PKC
Kinase Assay--
For
immunoprecipitation kinase assay of PKC
, K562 and HL60 cells were
lysed in RIPA lysis buffer (150 mM NaCl, 1% Nonidet P-40,
0.5% deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) at
4 °C in the presence of 1 mM phenylmethylsulfonyl
fluoride and 20 µg/ml leupeptin. Lysates were precleared by
incubation with 50 µl of protein A-Sepharose beads (Sigma) for 1 h at 4 °C. PKC
antibody was first bound to 50 µl of protein
A-Sepharose at 4 °C for 1 h prior to addition to clarified
lysates and further incubation at 4 °C for 1 h.
Immunoprecipitates were washed once with RIPA buffer and once with
kinase assay buffer (50 mM Tris, pH 7.5, 10 mM
MgCl2, 0.5 mM EGTA, 0.1 mM
CaCl2) to remove unbound proteins. Washed beads were
resuspended in kinase buffer containing 40 µg/ml phosphatidylserine,
10 µg of histone substrate, 10 µM ATP, and 10 µCi of
[
-32P]ATP. The reactions were incubated at room
temperature for 30 min and terminated by addition of SDS buffer and
boiling prior to SDS-polyacrylamide gel electrophoresis and autoradiography.
Generation and Use of Recombinant
Adenoviruses--
Constitutively active PKC
was generated by PCR
amplification of a fragment containing an A120E substitution within the
pseudosubstrate domain of human PKC
. Such a mutation has previously
been shown to induce constitutive activity in PKC
(21). The
EcoRV/NdeI-digested fragment was subcloned into
similarly digested full-length PKC
cDNA and sequenced to confirm
the presence of this single mutation. PKC
A120E (designated caPKC
)
was then subcloned into pXCX.CMV, a transfer vector based on pXCX2 (22)
containing the expression cassette from pRcCMV (Stratagene).
Recombinant adenoviruses containing either the green fluorescent
protein (GFP) (a kind gift from Phil Poronik, University of Sydney,
NSW, Australia) or constitutively active PKC
were produced by
homologous recombination with pJM17 (a circular form of the adenovirus
genome) in HEK 293 cells as described (23). Once recombinant viruses
were plaque purified and characterized by Western blot analysis,
concentrated viral lysates were generated by infection of HEK 293 cells
as described (23). Viral lysates (titers of ~1012
plaque-forming unit/ml) were used to infect K562 cells. Briefly, viral
lysate was transfected into K562 cells using the LipofectAMINE reagent
(Life Technologies, Inc.). After 24 h, cells were treated with
taxol and/or AG957 for 24 h, at which time cells were assayed for
apoptosis as described above. Infection efficiency was routinely >95%
as judged by fluorescence microscopy for GFP expression. caPKC
expression was confirmed by immunoprecipitation kinase assay of total
cell lysates from cells 24 h after infection as described above.
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RESULTS |
K562 Chronic Myelogenous Leukemia Cells Are Resistant to
Taxol-induced Apoptosis--
Recently, we demonstrated that PKC
expression is important for resistance of K562 cells to apoptotic
agents including taxol (19). Specifically, overexpression of PKC
leads to enhanced resistance to taxol-induced apoptosis, whereas
antisense inhibition of PKC
expression leads to sensitization to
taxol-induced apoptosis (19). These results suggested that changes in
PKC
expression and/or activity may play a critical role in the
survival of K562 cells to apoptotic stress. To determine whether the
level of PKC
expression is a key determinant of resistance to
drug-induced apoptosis, we studied two human leukemic cell lines that
differ in their sensitivity to taxol-induced apoptosis. HL60 cells are sensitive to taxol, undergoing dose-dependent apoptosis
such that in the presence of 100 nM taxol, >90% of HL60
cells are apoptotic after 24 h (Fig.
1A). In contrast, K562 cells
are highly resistant to taxol-induced apoptosis showing little or no
apoptosis after 24 h in up to 325 nM taxol (Fig.
1B).

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Fig. 1.
K562 chronic myelogenous leukemia cells are
resistant to taxol-induced apoptosis. K562 (A) and HL60
(B) leukemia cells were incubated with the indicated
concentration of taxol for 24 h and assessed for apoptosis as
described under "Experimental Procedures." Results are plotted as
percentages of normal or apoptotic cells.
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PKC
Is Activated in Response to Taxol in K562 but Not HL60
Cells--
To assess whether this difference in resistance to
taxol-induced apoptosis correlates with PKC
expression, both cell
lines were treated with 100 nM taxol for up to 50 h,
and total cell lysates were subjected to immunoblot analysis for PKC
as described previously (19) (Fig. 2).
Interestingly, these cell lines express similar basal levels of PKC
(Fig. 2, A and B). Furthermore, PKC
levels do not vary in response
to taxol-induced apoptotic stress, suggesting that differences in
resistance to apoptosis are not due to differences in PKC
expression. Therefore, we measured PKC
activity in K562 and HL60
cells during taxol-induced apoptotic stress (Fig. 2C). Cells
were treated with 100 nM taxol and PKC
immunoprecipitated from cell lysates at the indicated times and assayed
for PKC
activity as described under "Experimental Procedures." Immunoblot analysis demonstrated that >90% of total cellular PKC
is captured by this antibody (data not shown). Basal PKC
activity is
similar in K562 and HL60 cells prior to exposure to taxol, consistent
with the similar levels of PKC
expression in these cells. When HL60
cells are treated with 100 nM taxol, very little change in
PKC
activity is observed over the first 25 h, by which time
>90% of HL60 cells are apoptotic. After 25 h, PKC
activity drops to undetectable levels by 36 h. This loss of PKC
activity is not due to proteolytic degradation because the levels of PKC
do
not change over this time course (Fig. 2A).

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Fig. 2.
Immunoblot and activity analysis of
PKC in K562 and HL60 cells during
taxol-induced apoptotic stress. K562 (A) and HL60
(B) cells were incubated with 100 nM taxol, and
at the indicated times cell lysates were prepared and subjected to
immunoblot analysis for PKC as described under "Experimental
Procedures." C, K562 (K) and HL60
(H) cells were treated with taxol as described above, and at
the indicated times cell lysates were prepared and subjected to
immunoprecipitation and PKC kinase analysis using histone H1 as
substrate as described under "Experimental Procedures."
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In contrast, PKC
activity in K562 cells is dramatically induced in
response to taxol (Fig. 2C). Increased PKC
activity is seen at 6 h, peaks at 12 h, and is sustained over the
subsequent 36 h. Quantitation of the data indicates a 6.5-fold
increase in PKC
activity by 12 h. These data demonstrate that
endogenous PKC
is activated in response to apoptotic stress in K562
cells but not HL60 cells, suggesting that sustained PKC
activation may be important in the resistance of K562 cells to taxol-induced apoptosis.
Bcr-Abl Is Involved in PKC
Activation during Taxol-induced
Apoptotic Stress--
The Bcr-Abl tyrosine kinase is the oncogene
responsible for CML and is highly expressed in K562 cells but is absent
from HL60 cells. Bcr-Abl is required for both transformation and
resistance to drug-induced apoptosis of K562 cells (5, 10, 11).
However, the proximal downstream targets of Bcr-Abl critical for the
resistance phenotype are not known. Given the involvement of PKC
activation in taxol resistance, we determined whether taxol-induced
PKC
activation in K562 cells was dependent upon Bcr-Abl kinase
activity. Furthermore, because Bcr-Abl can directly bind,
phosphorylate, and activate PI 3-kinase (24, 25) to generate PI
3,4,5-trisphosphate, which can in turn activate PKC
(26), we also
determined whether PI 3-kinase activity was required for taxol-induced
PKC
activation. K562 cells were treated with 100 nM
taxol in the presence or absence of the Bcr-Abl inhibitor tyrphostin
AG957 (12, 20), or the PI 3-kinase inhibitors wortmannin (27) or
LY294002 (28) for 12 h. PKC
activity was subsequently
determined by immunoprecipitation kinase assay. Taxol treatment leads
to potent activation of PKC
above the basal activity seen in the
absence of taxol (Fig. 3A). However, tyrphostin AG957 completely abolishes taxol-induced
PKC
activation, whereas neither wortmannin (100 nM) nor
LY294002 (100 µM) affected PKC
activation despite the
fact that they were used at concentrations well above their
IC50 values (5 nM for wortmannin (27) and 1.4 µM for LY294002 (28)). Tyrphostin AG957-mediated inhibition of taxol-induced PKC
activation is
dose-dependent and is nearly complete at 30 µM consistent with the effective doses of the compound
that inhibit Bcr-Abl activity (12, 20). Furthermore, tyrphostin AG957
leads to dose-dependent sensitization of K562 cells to
taxol-induced apoptosis consistent with its ability to inhibit PKC
activation (Fig. 3C). Neither wortmannin nor LY294002 had
any effect on taxol-induced apoptosis, consistent with the lack of
inhibition of PKC
observed with these PI 3-kinase inhibitors. Taken
together, these data indicate that Bcr-Abl is an upstream regulator of
taxol-induced PKC
activation, that PKC
activation is important
for resistance of K562 cells to taxol, and that Bcr-Abl-mediated PKC
activation does not require PI 3-kinase activity.

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Fig. 3.
Tyrphostin AG957 inhibits taxol-induced
PKC activation and sensitizes K562 cells to
taxol-induced apoptosis. K562 cells were treated with 100 nM taxol in the absence or presence of 100 µM
LY294002, 100 nM wortmannin, and tyrphostin AG957 at the
indicated concentrations. At 12 h, cell lysates were prepared and
subjected to immunoprecipitation PKC kinase and immunoblot analysis
(A and B) and for measurement of apoptosis
(C) as described under "Experimental Procedures."
White bars, nonapoptotic; cross-hatched bars,
apoptotic.
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Tyrphostin AG957-blocked K562 Cells Can Be Rescued from
Taxol-induced Apoptosis by Constitutively Active PKC
--
To
determine whether PKC
activity is sufficient to protect K562 cells
from taxol-induced apoptosis, we used recombinant adenovirus gene
transfer to express constitutively active PKC
in K562 cells. Constitutively active PKC
adenovirus (caPKC
-Ad) was produced by
mutation of alanine 120 to glutamic acid in the pseudosubstrate region
of PKC
as described under "Experimental Procedures." This mutation has been demonstrated to lead to constitutive activation of
PKC
(21). K562 cells were infected with either GFP or caPKC
adenovirus for 24 h. GFP immunofluorescence demonstrated that >95% of cells are infected under these conditions.
Immunoprecipitation kinase assays demonstrate that GFP-Ad-infected
cells contain PKC
activity comparable with uninfected cells (Fig.
4A). In contrast, cells
infected with caPKC
-Ad contain 3-5-fold higher levels of PKC
activity. Adenovirus-infected cells were next treated with taxol and
tyrphostin AG957 for 24 h, and the level of apoptosis was assessed
(Fig. 4B). Neither taxol nor tyrphostin AG957 alone induced
apoptosis in GFP- or caPKC
-expressing K562 cells. However, in the
presence of tyrphostin AG957, taxol induced apoptosis in GFP-expressing
cells. In contrast, expression of caPKC
blocked taxol-induced
apoptosis in tyrphostin AG 957-treated cells. These results demonstrate
that PKC
is a downstream target of Bcr-Abl and that PKC
activity
is sufficient to mediate the anti-apoptotic effects of Bcr-Abl.

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Fig. 4.
Expression of constitutively active
PKC rescues AG957-treated K562 cells from
taxol-induced apoptosis. A, K562 cells were infected
with constitutively active (CA-PKC -Ad) or GFP-expressing
adenovirus for 48 h. Infected cells were lysed and cellular PKC
immunoprecipitated and assessed for kinase activity as described under
"Experimental Procedures." B, K562 cells were infected
with constitutively active PKC (aPKC ) or
GFP-expressing adenovirus as described for A. 24 h
after infection, cells were treated with taxol and/or tyrphostin AG957
for a further 24 h as indicated. Cells were fixed and scored for
apoptosis as described under "Experimental Procedures."
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DISCUSSION |
PKC is a family of serine/threonine, lipid-dependent
kinases implicated in the regulation of cellular proliferation,
differentiation, and apoptosis (19, 29-32). We have characterized the
function of the three PKC isozymes expressed in K562 cells, PKC
,
II, and
, through selective overexpression and
inhibition of expression of each isozyme (19, 31-33). Overexpression
of PKC
leads to gene dose-dependent cytostasis and
increased sensitivity to PMA-induced megakaryocytic differentiation,
whereas inhibition of PKC
expression blocks PMA-induced
differentiation, demonstrating that PKC
plays a direct role in this
process (31). In contrast, overexpression of PKC
II leads
to a small increase in proliferation and resistance to PMA-induced
differentiation (31). Inhibition of PKC
II expression blocks proliferation, demonstrating that PKC
II is
required for this process (31). The proliferative effects of
PKC
II are mediated, at least in part, through the cell
cycle-regulated translocation and activation of the enzyme at the
nucleus (32, 33). Nuclear PKC
II directly phosphorylates
the nuclear envelope protein lamin B at sites involved in mitotic
nuclear lamina disassembly and cell cycle progression through the
G2/M phase (33-35).
Atypical PKC
plays no obvious role in K562 cell proliferation or
differentiation (19). Rather, overexpression of PKC
leads to
enhanced resistance to drug-induced apoptosis, and antisense inhibition
of PKC
expression leads to increased sensitivity to apoptosis,
indicating an important role in resistance to drug-induced apoptosis
(19). Atypical PKC isozymes can also protect NIH 3T3 cells against
UV-induced apoptosis (21), indicating that atypical PKCs may play a
general role in cell survival and protection from apoptotic stress.
However, the mechanism by which PKC
protects cells from apoptosis is
not known. In NIH 3T3 cells, atypical PKC activity is inhibited by UV
exposure prior to the onset of apoptosis, suggesting that atypical PKC
activity is required for cell survival (21). In this and our previous
studies, we provide direct evidence that PKC
activity is both
necessary and sufficient for resistance of K562 cells to drug-induced apoptosis.
Our results with PKC
are reminiscent of those described previously
for Bcr-Abl, whose expression and tyrosine kinase activity is required
for resistance of K562 cells to drug-induced apoptosis (5, 10, 11-15).
Indeed, although both HL60 and K562 cells express similar levels of
PKC
, the pattern of PKC
activity in these cells during apoptotic
stress is distinct. During apoptotic stress, sustained activation of
PKC
is observed in K562 cells but not HL60 cells, suggesting that
K562 cells possess an upstream regulator of PKC
activity that is
either not present or is defective in HL60 cells. Furthermore, we find
that inhibition of Bcr-Abl blocks taxol-induced PKC
activation and
induces apoptosis in K562 cells, demonstrating that Bcr-Abl is required
for taxol-induced PKC
activation. An interesting aspect of our
results is the fact that PI 3-kinase activity is not required for
Bcr-Abl-mediated PKC
activation or protection from taxol-induced
apoptosis. PI 3,4,5-trisphosphate can stimulate atypical PKC activity
in vivo (24). Our results do not preclude involvement of the
PI 3-kinase pathway in the activation of PKC
in response to other
cellular stimuli.
Bcr-Abl is a well characterized oncoprotein whose transforming
phenotype is linked to its anti-apoptotic properties. Inhibition of
Bcr-Abl activity with the tyrosine kinase inhibitors quercetin or
genistein (15) or Bcr-Abl expression with antisense oligonucleotides against Bcr-Abl (16, 17) sensitizes K562 cells to apoptosis. Our
results using the highly selective Bcr-Abl inhibitor tyrphostin AG957
are consistent with these studies and demonstrate that PKC
activation is a requisite downstream target of Bcr-Abl necessary for
its anti-apoptotic function. Future studies will focus on determining
the molecular mechanism by which Bcr-Abl activates PKC
and on
identification of the downstream targets of PKC
that mediate its
anti-apoptotic effects.