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Volume 272, Number 50, Issue of December 12, 1997
pp. 31321-31325
(Received for publication, August 5, 1997)
From the Laboratory of Molecular Pharmacology, Division of Basic
Sciences, NCI, National Institutes of Health, Bethesda, Maryland
20892
ABSTRACT We previously demonstrated that the anticancer
agent and protein kinase C (PKC) inhibitor 7-hydroxystaurosporine
(UCN-01) induces apoptosis independently of p53 and protein synthesis
in HL60 cells. We now report the associated changes of PKC isoforms. PKC INTRODUCTION Protein kinase C (PKC)1
is a key enzyme for transduction of extracellular and
phospholipid-mediated signals as well as for tumor promotion (1). The
biochemical mechanisms underlying PKC action have become increasingly
clear (1-3). PKC isoforms have been categorized in three subclasses:
conventional PKCs (cPKCs) ( In the present study, we investigated PKC isoform activities during
UCN-01-induced apoptosis in human myeloid leukemia HL60 cells. HL60
cells were chosen for these studies because they undergo rapid
apoptosis to various agents including UCN-01. This apoptosis is
p53-independent as HL60 cells are p53 null (22) and does not require
protein synthesis (14, 23). It is preceded by transient activation of
cyclin B1/Cdc2 kinase (14, 24). The changes of PKC activity were also
examined in HL60 cells undergoing apoptosis after treatment with the
topoisomerase inhibitors, CPT and etoposide (VP-16). We found that
UCN-01 inhibited PKC MATERIALS AND METHODS UCN-01 was provided by Dr.
H. Nakano (Kyowa Hakko Co., Japan) or the Drug Synthesis and Chemistry
Branch, NCI, National Institutes of Health. CPT was provided by Dr.
M. R. Wall (Research Triangle Institute, Research Triangle Park,
NC) or the Drug Synthesis and Chemistry Branch, NCI, National
Institutes of Health.
N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethylketone (z-VAD-fmk) was purchased from Enzyme System Products (Dublin, CA).
Other drugs and reagents, unless otherwise mentioned, were purchased
from Sigma.
Anti-PKC antibodies were from Santa Cruz Biotechnology (Santa Cruz,
CA). Anti-rabbit Ig horseradish peroxidase antibody was purchased from
Amersham Life Science, Inc. (Arlington Heights, IL).
[14-C]thymidine (53.6 mCi/mmol),
[ Human promyelocytic leukemia HL60 cells were
grown at 37 °C in the presence of 5% CO2 in RPMI 1640 medium supplemented with 10% fetal bovine serum (Life Technologies,
Inc., Gaithersburg, MD), 2 mM glutamine, 100 units/ml
penicillin, and 100 µg/ml streptomycin.
Cytosols from untreated
control and treated HL60 cells were extracted using nucleus buffer (150 mM NaCl, 1 mM KH2PO4, 5 mM MgCl2, 1 mM EGTA, 0.1 mM p-aminoethylbenzenesulfonylfluoride, 0.15 units/ml aprotinin, 1.0 mM Na3VO4,
5 mM HEPES (pH 7.4), 10% glycerol, and 0.3% Triton X-100)
as described previously (25). Protein concentration was measured using
a protein assay kit according to the manufacturer instructions
(Bio-Rad).
Cytosols (50 µl)
from control or treated (1 × 106) HL60 cells were
mixed with 50 µl of 2 × reaction buffer containing 5 mM HEPES (pH 7.4), 5 mM MgCl2, 10 µM ATP, 40 µg/ml phosphatidylserine, 3.3 µM dioleoylglycerol, 1.2 mM
CaCl2, 25 µM MBP4-14, 2 µCi
[ 350-µl cytosols from control
or treated (2 × 107) HL60 cells were precleared with
20 µl of protein A-Sepharose for 1 h and incubated with anti-PKC
antibodies and 30 µl of protein A-Sepharose at 4 °C for 4 h.
The beads were washed three times with nucleus buffer. This was
followed by 2 washes with kinase buffer (50 mM Tris-HCl (pH
7.4), 10 mM NaF, 1 mM
Na3VO4, 0.5 mM EGTA, 0.5 mM EDTA, 2 mM MgCl2, 5 µg/ml
leupeptin, 1 mM phenylmethylsulfonyl fluoride).
Immunocomplex beads were obtained by incubating
different anti-PKC antibodies with cytosolic extracts in 20 µl of
reaction buffer containing 20 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 10 µM cold ATP, 0.4 mg/ml histone H1, 40 µg/ml PS, 3.3 µM dioleoylglycerol, 5 µCi of [ Cytosolic samples were
electrophoresed at 120 V on 12% SDS-PAGE gels and electrophoretically
transferred to Immobilon membranes (Millipore, Bedford, MA) for 2 h at 30 V. The membranes were blocked overnight in phosphate-buffered
saline-Tween 20 (PBS-T) containing 5% non-fat dried milk. Probing with
selected anti-PKC antibodies (1 µg/ml in PBS-T) for 1 h was
followed by incubation with horseradish peroxidase-labeled anti-rabbit
IgG (1:1000 dilution). After washing in PBS-T, membranes were developed
using the enhanced chemiluminescence (ECL) detection system (NEN Life
Science Products).
HL60 cells were spun and
resuspended at 106 cells/ml in phosphate-free RPMI 1640 medium supplemented with 10% dialyzed fetal bovine serum once. After
1 h, 50 µCi/ml [32P]orthophosphate was added to
cell cultures for 1 h. Then cells were chased in isotope-free
medium for 1 h prior to UCN-01 treatment. Cytosol extracts were
boiled in electrophoresis sample buffer and analyzed by 10% SDS-PAGE
gels. Gels were dried and autoradiographed at DNA fragmentation related to apoptosis was measured by
filter elution assay as described previously (8, 25).
RESULTS Our previous studies indicated that UCN-01
and CPT are potent inducers of apoptosis in HL60 cells (14, 27).
After 3 h of drug treatment, 60-90% of the cells exhibit
apoptosis with typical morphological changes and internucleosomal
fragmentation. To investigate whether PKC was involved in apoptosis
induced by these anticancer drugs, we treated HL60 cells with UCN-01 or
CPT and extracted cytosol and membrane fractions at various times. The
synthetic peptide, MBP4-14, was used as a specific and sensitive substrate to assay PKC activity (26). We found that phosphorylation of
MBP4-14 first decreased during the first hour of UCN-01 treatment compared with control and then increased progressively above control values 3 h after the beginning of treatment. CPT increased the phosphorylation of MBP4-14 already after 1 h of treatment (Fig. 1). These measurements were performed in
cytosolic fractions. In membrane fractions, there was no significant
difference between basal and stimulated levels of MBP4-14
phosphorylation and no change of MBP4-14 phosphorylation after
exposure to UCN-01 or CPT for 3 h (not shown). These data indicate
that PKC activity is induced by UCN-01 and CPT treatment in HL60 cells
undergoing apoptosis.
[View Larger Version of this Image (18K GIF file)]
The above data raised the question as to what
kinds of PKC isoforms were activated by UCN-01 or CPT. MBP4-14 is
selective for PKC
[View Larger Version of this Image (57K GIF file)]
Caspases (ICE-like proteases) are key mediators of
apoptosis (28-30), and we previously showed that the caspase inhibitor
z-VAD-fmk prevents UCN-01-induced apoptosis in HL60 cells (14). Fig.
3A, shows that z-VAD-fmk also
prevents apoptotic DNA fragmentation induced by CPT in HL60 cells.
However, z-VAD-fmk does not inhibit the DNA fragmentation induced by
UCN-01- and CPT-activated cytosol in cell-free system (8, 14, 25) (Fig.
3B). Under these conditions, we examined whether z-VAD-fmk
affected the PKC changes induced by UCN-01 or CPT in HL60 cells
undergoing apoptosis. We found that z-VAD-fmk blocked the activation of
PKC
[View Larger Version of this Image (18K GIF file)]
[View Larger Version of this Image (23K GIF file)]
To further investigate the
mechanisms by which UCN-01 or CPT regulate PKC
[View Larger Version of this Image (32K GIF file)]
We next examined whether the activation of PKC
[View Larger Version of this Image (35K GIF file)]
Since neither proteolytic activation nor change in protein levels
appear to account for PKC
[View Larger Version of this Image (31K GIF file)]
[View Larger Version of this Image (16K GIF file)]
DISCUSSION The results of Fig. 1 show that although UCN-01 is a PKC inhibitor
and CPT is a topoisomerase I inhibitor, both of them induced activation
of PKC. Using immunoprecipitation assay for PKC activities, we found
that UCN-01 inhibited PKC Caspases play a central role in the apoptosis pathways (28-30).
z-VAD-fmk, a cell-permeable caspase inhibitor with broad specificity, blocks apoptosis induced by various stimuli including treatment by
UCN-01 or CPT. Our results demonstrate that z-VAD-fmk selectively inhibited the activation of PKC Our data (Fig. 5) rule out that UCN-01, CPT, or z-VAD-fmk directly
affected PKC The mechanisms (pathways) by which UCN-01 and CPT induce
phosphorylation and activation of PKC FOOTNOTES ACKNOWLEDGEMENT We thank Dr. Kurt W. Kohn for discussion and
support during the course of these studies.
REFERENCES
Activation of PKC
Downstream from Caspases during Apoptosis
Induced by 7-Hydroxystaurosporine or the Topoisomerase Inhibitors,
Camptothecin and Etoposide, in Human Myeloid Leukemia HL60 Cells*
, 
I, II, 
, and 
activities were measured after
immunoprecipitation of cytosols from UCN-01-treated HL60 cells. UCN-01
had no effect on PKC and inhibited kinase activity of PKCI,
II, and . PKC activity was initially inhibited at 1 h,
and subsequently increased as cells underwent apoptosis 3 h after
the beginning of UCN-01 treatment. Camptothecin (CPT) and etoposide
(VP-16) also markedly enhanced PKC activity during apoptosis in HL60
cells. However, CPT did not affect PKCI, II and , and
activated PKC. PKC activation was not due to increased protein
levels or proteolytic cleavage but was associated with PKC
autophosphorylation in vitro and increased phosphorylation
in vivo. We also found that not only PKC but also PKC
I was proteolytically activated in HL60 cells during apoptosis. The
PKC activation and hyperphosphorylation were abrogated by
N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethylketone (z-VAD-fmk) under conditions that abrogated apoptosis. z-VAD-fmk also prevented PKC and I proteolytic activation. Together these findings suggest that caspases regulate PKC activity during apoptosis in HL60 cells. At least two modes of activation were observed: hyperphosphorylation for PKC and proteolytic activation for PKC and I.
, I, II, and 
) which require
phosphotidylserine (PS), diacylglycerol (DAG); and Ca2+;
novel PKCs (nPKCs) (, 
, 
, and 
) which require PS and DAG but not Ca2+, and the atypical PKCs (aPKCs) (, 
, and
) (3). Several observations suggest the implication of PKC in
apoptosis. For example, it has been observed that the activation of PKC
by exposure to phorbol 12-myristate 13-acetate (PMA) either alone or in
conjunction with Ca2+ ionophore induces apoptosis in
lymphoid (4-6) and myeloid (7) cells. PMA suppresses steroid-induced
apoptosis in thymic lymphocytes (8) and opposes topoisomerase-induced
apoptosis in HL60 cells as these cells differentiate (7). Several PKC
inhibitors have been reported to induce apoptosis. Staurosporine is
one of the most potent and universal inducers of apoptosis in a variety
of cell lines (9). Its 7-hydroxy derivative, UCN-01, which is a more
specific PKC inhibitor (10) and an effective antitumor agent (11, 12),
also induces apoptosis in T lymphoblasts (13), K562, HT29 cells, and
HL60 cells independently of p53 (14). UCN-01-induced apoptosis in HL60
and K562 cells is independent of protein synthesis (14). Calphostin C
induces apoptosis in four human glioma cell lines (GB-1, T98G, U-373MG,
and A-172) (15) and HL60 cells (16). Additional evidence for PKC
activation during apoptosis is that ceramide activates PKC and
down-regulates PKC during apoptosis (17-19). Emoto et al.
(20) reported proteolytic activation of PKC during apoptosis
induced by DNA damaging agents (ionizing radiation, camptothecin (CPT)
and 1-[-D-arabinofuranosyl]cytosine (ara-C)) in human
myeloid leukemia U937 cells (21).
I, II, and activity in drug-treated cells
as well as in vitro. For PKC, UCN-01 first inhibited the
kinase activity and then activated PKC. CPT-induced apoptosis
was associated with marked increase in PKC and activities. The
mechanisms of PKC activation and their relationship to caspases
(ICE-like proteases) are investigated.
-32P]ATP (4500 Ci/mmol), and
[32P]orthophosphate were purchased from NEN Life Science
Products (Boston, MA). Protein A-Sepharose 4B was from Pharmacia
Biotech Inc. (Sweden). P-81 ion exchange filter paper was obtained from Whatman.
-32P]ATP. After incubation for 7 min at 30 °C, the
transfer of [32P]phosphate was quantified by the
phosphocellulose paper-binding method (26).
-32P]ATP in the absence or presence of 1.2 mM CaCl2 depending on the PKC isoform activity
measured. Incubations were carried out at 30 °C for 10 min. Samples
were loaded onto 12% SDS-PAGE gels (NOVEX, San Diego, CA) and
electrophoresed at 120 V for 2 h. For quantification of kinase
activity in immunoprecipitates, gels were dried, and the extent of
histone H1 phosphorylation was measured using a PhosphorImager
(Molecular Dynamics, Sunnyvale, CA).
Phosphorylation in Cells
70 °C.
Fig. 1.
PKC activity changes in HL60 cells treated
with UCN-01 or CPT. Cytosol was prepared from untreated cells or
from cells treated with 10 µM UCN-01 or 1 µM CPT for the indicated times. Phosphorylation of
MBP4-14 was performed in a mixture containing reaction buffer and
cytosol fraction.
Activity
, I, II, and isoforms but has been
reported to be a poor substrate for other isoforms (26). From the work
by Seynaeve et al. (10), we also know that UCN-01 inhibits
PKC, , , , and and does not inhibit PKC in
vitro. Since PKC was undetectable in HL60 cells and PKC was
expressed only very weakly using Western blotting (data not shown) (1),
we used anti-PKC, I, II, , and antibodies and
immunoprecipitation to determine the effects of UCN-01 and CPT on PKC
isoform activities. Fig. 2 shows that UCN-01 inhibited all the PKC isoforms tested at 1 h of treatment, which is consistent with the known anti-PKC activity of UCN-01 in
vitro (10). However, at 3 h, PKC activity was markedly
stimulated (5-fold) by UCN-01. CPT increased PKC and activities
but had no effects on PKCI and II activities. Neither UCN-01 nor
CPT affected PKC activity (data not shown). These data indicate that apoptosis induced by UCN-01 and CPT treatment is associated with marked
PKC activation.
Fig. 2.
PKC isoform changes in HL60 cells treated
with UCN-01 or CPT. Cytosol from untreated HL60 cells or cells
treated with 10 µM UCN-01 or 1 µM CPT were
incubated with the indicated antibodies (left) for 2 h
at 4 °C. Immunoprecipitates (IP) were collected by
absorption with protein G-Sepharose for another 1-h incubation at
4 °C. Protein G-Sepharose beads were first washed with nucleus buffer and after with kinase buffer. Histone H1 kinase reactions were
carried out at 30 °C for 10 min and were terminated by adding SDS-PAGE sample buffer. Proteins were separated by 12% SDS-PAGE. Gels
were dried and exposed in PhosphorImager cassettes.
Activation
induced either by UCN-01 or CPT. However, z-VAD-fmk did not
suppress the CPT-induced activation of PKC (Fig.
4A) and had no effects on the
inhibitions of PKCI, II, and activities induced by UCN-01
(Fig. 4). We next tested whether PKC was activated in response to
the topoisomerase II inhibitor, VP-16. We found that VP-16 also
activated PKC in HL60 cells undergoing apoptosis (8) (Fig.
4C). These data suggested that apoptosis induced by
different pathways (topoisomerase inhibition for CPT or VP-16 or
protein kinase inhibition for UCN-01) is associated with PKC
activation in HL60 cells. The observation that z-VAD-fmk blocked PKC
activation suggests that PKC is downstream from caspases during
apoptosis in HL60 cells.
Fig. 3.
Inhibition of CPT and UCN-01-induced DNA
fragmentation by z-VAD-fmk. A, HL60 cells were treated with
10 µM UCN-01 or 1 µM CPT in the absence or
presence of 50 µM z-VAD-fmk for 3 h before
measurement of DNA fragmentation by filter elution assay. B,
cytosols were prepared from untreated HL60 cells (c-cyto) or from cells treated with 10 µM UCN-01
(UCN-cyto) or 1 µM CPT (CPT-cyto) for 3 h. Incubations with nuclei isolated from untreated cells were performed in the absence or presence of 50 µM
z-VAD-fmk at 37 °C for 30 min.
Fig. 4.
Effects of UCN-01, CPT, VP-16, and z-VAD-fmk
on PKC activity in HL60 cells. HL60 cells were exposed to
either 10 µM UCN-01 or 1 µM CPT in the
absence or presence of 50 µM z-VAD-fmk for 3 h
(panels A and B). HL60 cells were treated with 50 µM VP-16 for the indicated times (panel C).
Cytosol fractions from untreated or treated cells were
immunoprecipitated with the indicated anti-PKC antibodies
(panel A) or anti-PKC and antibodies (panels
B and C). PKC activity was measured using histone H1 as
substrate.
Activation Is Not Due to a Direct Effect of UCN-01
or CPT, and Suppression of PKC Activation Is Not Due to a Direct
Effect of z-VAD-fmk on PKC Activity
activity, we first
tested whether UCN-01 or CPT directly affected PKC activity using
in vitro kinase assays. PKC immunoprecipitates obtained
from control cytosol were tested for kinase activity with UCN-01 or
CPT. Fig. 5A shows that UCN-01 did not activate but rather inhibited PKC activity directly. CPT had
no direct effect on PKC activity (Fig. 5A). z-VAD-fmk was
also tested in this system and did not exhibit a direct effect on
PKC activation induced by UCN-01 or CPT (Fig. 5B). These
results indicated that the PKC changes observed in cytosols from cells treated with UCN-01 or CPT in the absence or presence of z-VAD-fmk were
indirect.
Fig. 5.
In vitro effects of UCN-01, CPT, and
z-VAD-fmk on PKC activity. A, cytosols from untreated
HL60 cells were immunoprecipitated with anti-PKC antibody. UCN-01 or
CPT was added to the immunoprecipitates. Then histone H1 kinase
activity was measured. B, cytosol was prepared from
untreated cells or from cells treated with CPT or UCN-01 for 3 h.
Cytosols were then incubated with 50 µM z-VAD-fmk for 30 min at 30 °C. Samples were immunoprecipitated with anti-PKC antibody. Histone H1 kinase activity was measured. DMSO,
dimethyl sulfoxide.
by UCN-01 or CPT and
the effects of z-VAD-fmk might be due to alterations of the PKC
protein. Western blot analyses were carried out on cytosols from
untreated, UCN-01- or CPT-treated HL60 cells using anti-PKC
antibody. PKC protein levels remained unchanged after drug-treatments (Fig. 6A).
Thus, PKC activation by UCN-01 and CPT was not due to changes in
PKC protein levels. We also measured the other PKC isoforms. Protein
levels of PKCI, II, and did not change significantly (Fig. 6,
B-D). Interestingly, UCN-01 and CPT induced
PKCI and cleavages when HL60 cells underwent apoptosis, and
z-VAD-fmk blocked these proteolytic effects. Proteolytic activation of
PKC by caspases is consistent with results obtained in U-937 cells
(21). However, our data suggest that PKCI is also cleaved by
caspases during apoptosis in HL60 cells.
Fig. 6.
UCN-01- and CPT-induced apoptosis are
associated with proteolytic cleavage of PKCI and while PKC
and II proteins appear unchanged. HL60 cells were treated with
10 µM UCN-01 or 1 µM CPT in the absence and
presence of z-VAD-fmk. Cytosols from control and treated cells were
subjected to Western blotting analysis using antibodies against PKC,
I, II, and (panels A, B, C, and D,
respectively).
activation, we next tested the effects of
UCN-01 and CPT on the PKC autophosphorylation. It is indeed known
that autophosphorylation is one of the important modes of regulation of
PKC activity (31). Autophosphorylation assays for PKC were carried
out essentially as described for histone H1 phosphorylation except that
histone H1 was omitted from the reactions. Fig.
7A shows that treatment of
HL60 cells for 3 h with either UCN-01 or CPT increased PKC
autophosphorylation (Fig. 7A) and that z-VAD-fmk blocked
this increased autophosphorylation (Fig. 7B). Finally, we
performed experiments to test in vivo phosphorylation of
PKC in HL60 cells treated with UCN-01. Fig.
8 shows that UCN-01 increased PKC
phosphorylation after 1 h of treatment and that z-VAD-fmk
inhibited the UCN-01-induced PKC phosphorylation. These results
suggest that caspase activation during apoptosis in HL60 cells is
upstream from PKC autophosphorylation and activation.
Fig. 7.
UCN-01- and CPT-induced apoptosis increase
PKC autophosphorylation in vitro. A, PKC
autophosphorylation in UCN-01- and CPT-activated cytosols was carried
out essentially as described for histone H1 phosphorylation except that
histone H1 was omitted. B, treatment of cells with z-VAD-fmk
inhibits PKC autophosphorylation induced by UCN-01 or CPT.
Fig. 8.
UCN-01-induced apoptosis is associated with
PKC phosphorylation in vivo. HL60 cells were
incubated in phosphate-free RPMI 1640 medium supplemented with 10%
dialyzed fetal bovine serum for 1 h. Fifty µCi/ml
[32P]orthophosphate was then added for 1 h. Cells
were washed once with phosphate-free medium for 1 h and then 10 µM UCN-01 was added. Cytosols were prepared at the
indicated times and were immunoprecipitated with anti-PKC antibody.
The beads were boiled in sample buffer and analyzed by SDS-PAGE gel
electrophoresis. The gel was dried and exposed to Kodak Rx film at
70 °C.
I, II, and activities in whole cells
as well as in vitro but that PKC was activated in HL60
cells undergoing apoptosis. Apoptosis induced by CPT, a DNA topoisomerase I inhibitor was also associated with activation of PKC
as well as PKC activation.
induced by UCN-01 and CPT. On the
other hand, z-VAD-fmk did not affect the inhibition of PKCI, II,
and activities by UCN-01 and the activation of PKC by CPT. At
the same time, we found that z-VAD-fmk blocked the proteolytic cleavages of PKCI and PKC, suggesting that PKC cleavage is not
the only mechanism for PKC activation. For PKC, we found no
evidence that PKC activation is associated with proteolytic cleavage.
. PKC activation is not due to changes in PKC
protein levels since control and cells treated with UCN-01 or CPT in
the absence and presence of z-VAD-fmk had approximately equal amounts
of PKC protein as detected by Western blot. Phosphorylation has
emerged as an important mode of regulation for PKC (3, 32). For
example, mutation of Thr to Ala in PKC (Thr-497) results in an
inactive kinase (33), and PKC autophosphorylation has been shown to
regulate PKC activity by trans-phosphorylation at the activation loop
followed by autophosphorylation (31, 32). Our results show that UCN-01
and CPT activated autophosphorylation of PKC and that z-VAD-fmk
reversed such effects. Using [32P]orthophosphate
metabolic labeling, we also found increased phosphorylation of PKC
in HL60 cells undergoing apoptosis and blockade of these effects by
z-VAD-fmk. These findings suggest that alterations of PKC
phosphorylation might be important for modulating PKC activity and
might be regulated by caspases.
are yet unknown. We previously showed that both CPT and UCN-01 induce transient and unscheduled cyclin
B1/Cdc2 kinase activation before the onset of apoptosis in HL60 cells
treated with CPT or UCN-01 (14, 24). CPT and DNA damaging agents have
also been shown to activate other protein kinases including
DNA-dependent protein kinase (34), jun kinase (35, 36), and
c-Abl (36). Further investigation will be required to establish the
connections between activation of various protein kinases and caspases
during apoptosis and to establish the functional significance of PKC
activation in the apoptotic response of HL60 cells.
To whom correspondence should be addressed: Laboratory of
Molecular Pharmacology, Bldg. 37, Rm. 5C25, National
Institutes of Health, Bethesda, MD 20892-4255. Fax: 301-402-0752.
1
The abbreviations used are: PKC, protein
kinase C; UCN-01, 7-hydroxystaurosporine; CPT, camptothecin; VP-16,
etoposide; PS, phosphotidylserine; DAG, diacylglycerol; z-VAD-fmk,
N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethylketone; PAGE, polyacrylamide gel electrophoresis; PBS-T, phosphate-buffered saline-Tween 20.
Volume 272, Number 50,
Issue of December 12, 1997
pp. 31321-31325
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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M. E. Reyland, S. M. Anderson, A. A. Matassa, K. A. Barzen, and D. O. Quissell Protein Kinase C delta Is Essential for Etoposide-induced Apoptosis in Salivary Gland Acinar Cells J. Biol. Chem., July 2, 1999; 274(27): 19115 - 19123. [Abstract] [Full Text] [PDF]
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 A. Khwaja and L. Tatton Caspase-Mediated Proteolysis and Activation of Protein Kinase Cdelta Plays a Central Role in Neutrophil Apoptosis Blood, July 1, 1999; 94(1): 291 - 301. [Abstract] [Full Text] [PDF]
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M. F. Denning, Y. Wang, B. J. Nickoloff, and T. Wrone-Smith Protein Kinase Cdelta Is Activated by Caspase-dependent Proteolysis during Ultraviolet Radiation-induced Apoptosis of Human Keratinocytes J. Biol. Chem., November 6, 1998; 273(45): 29995 - 30002. [Abstract] [Full Text] [PDF]
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L. M. Martins, T. J. Kottke, S. H. Kaufmann, and W. C. Earnshaw Phosphorylated Forms of Activated Caspases Are Present in Cytosol From HL-60 Cells During Etoposide-Induced Apoptosis Blood, November 1, 1998; 92(9): 3042 - 3049. [Abstract] [Full Text] [PDF]
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T. Shimizu, C.-X. Cao, R.-G. Shao, and Y. Pommier Lamin B Phosphorylation by Protein Kinase Calpha and Proteolysis during Apoptosis in Human Leukemia HL60 Cells J. Biol. Chem., April 10, 1998; 273(15): 8669 - 8674. [Abstract] [Full Text] [PDF]
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F. Diana, R. Sgarra, G. Manfioletti, A. Rustighi, D. Poletto, M. T. Sciortino, A. Mastino, and V. Giancotti A Link between Apoptosis and Degree of Phosphorylation of High Mobility Group A1a Protein in Leukemic Cells J. Biol. Chem., March 30, 2001; 276(14): 11354 - 11361. [Abstract] [Full Text] [PDF]
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J. E. Kirby and D. M. Nekorchuk Bartonella-associated endothelial proliferation depends on inhibition of apoptosis PNAS, April 2, 2002; 99(7): 4656 - 4661. [Abstract] [Full Text] [PDF]
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ABSTRACT
