Phosphorylation of Bcl-2 Is a Marker of M Phase Events and
Not a Determinant of Apoptosis*
Yi-He
Ling
,
Carmen
Tornos§, and
Roman
Perez-Soler
¶
From the
Department of Thoracic/Head and Neck Medical
Oncology, Section of Experimental Therapy and § Department
of Pathology, The University of Texas M. D. Anderson Cancer
Center, Houston, Texas, 77030
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ABSTRACT |
Phosphorylation of Bcl-2 protein is a
post-translational modification of unclear functional consequences. We
studied the correlation between Bcl-2 phosphorylation, mitotic arrest,
and apoptosis induced by the anti-tubulin agent paclitaxel. Continuous
exposure of human cervical carcinoma HeLa cells to 50 ng/ml paclitaxel
resulted in mitotic arrest with a symmetrical bell-shaped curve over
time. The number of mitotic cells was highest at 24 h (82%), then
declined as arrested cells progressed into apoptosis, and barely no
mitotic cells were present at 48-60 h. The time curves of
paclitaxel-induced cyclin B1 accumulation and stimulation of
Cdc2/cyclin B1 kinase activity were identical and superimposable to
that of M phase arrest. In contrast, apoptosis was first detected at
12 h and steadily increased thereafter until the termination of
the experiments at 48-60 h, when about 80-96% of cells were
apoptotic. Bcl-2 phosphorylation was closely associated in time with M
phase arrest, accumulation of cyclin B1, and activation of Cdc2/cyclin
B1 kinase, but not with apoptosis. At 24 h, when about 82% of the
cells were in mitosis, almost all Bcl-2 protein was phosphorylated,
whereas at 48 h, when 70-90% of the cells were apoptotic, all
Bcl-2 protein was unphosphorylated. Similar results were obtained with
SKOV3 cells, indicating that the association of paclitaxel-induced M
phase arrest and Bcl-2 phosphorylation is not restricted to HeLa cells. We used short exposure to nocodazole and double thymidine to
synchronize HeLa cells and investigate the association of Bcl-2
phosphorylation with mitosis. These studies demonstrated that Bcl-2
phosphorylation occurs in tight association with the number of mitotic
cells in experimental conditions that do not lead to apoptosis.
However, a continuous exposure to nocodazole resulted in a pattern of
Bcl-2 phosphorylation, M phase arrest, and apoptosis similar to that observed with paclitaxel. The phosphatase inhibitor okadaic acid was
found to inhibit the dephosphorylation of phosphorylated Bcl-2 and to
delay the progression of nocodazole M phase-arrested cells into
interphase. In contrast, the serine/threonine kinase inhibitor staurosporine, but not the tyrosine kinase inhibitor genistein, led to
rapid dephosphorylation of phosphorylated Bcl-2 and accelerated the
progression of nocodazole M phase-arrested cells into interphase. Immune complex kinase assays in cell-free systems demonstrated that
Bcl-2 protein can be a substrate of Cdc2/cyclin B1 kinase isolated from
paclitaxel-treated cells arrested in M phase. Taken together, these
studies suggest that Bcl-2 phosphorylation is tightly associated with
mitotic arrest and fail to demonstrate that it is a determinant of
progression into apoptosis after mitotic arrest induced by anti-tubulin
agents.
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INTRODUCTION |
Apoptosis is controlled by a complex interplay between regulatory
proteins (1). Bcl-2, a 26-kDa integral membrane oncoprotein, was the
first anti-apoptosis gene product discovered (2). Several reports have
demonstrated that overexpression of Bcl-2 protein protects cells from
undergoing apoptosis in some cell systems, although a recent report
indicates that the level of this oncoprotein is not always correlated
with an increased ability of the cell to resist death-promoting stimuli
(3). Phosphorylation of Bcl-2
was first reported by Alnermri
et al. (4) in SF9 cells, and its functional implications
remain controversial. Haldar et al. (5, 6) recently reported
that treatment with either okadaic acid, a potent inhibitor of
phosphatase, or the anti-tubulin agent paclitaxel resulted in Bcl-2
protein phosphorylation and induction of programmed cell death in
lymphoid cells, suggesting that Bcl-2 phosphorylation may abrogate its
antiapoptotic function. Other studies, however, do not seem to support
this hypothesis. May et al. (7) demonstrated that treatment
of murine myeloid factor-dependent FDC-P1/ER cells with
interleukin-3 and bryostatin-1, a protein kinase C activator, resulted
in the induction of Bcl-2
protein hyperphosphorylation and
prevention of apoptosis, indicating that Bcl-2 protein phosphorylation
may not be associated with loss of function.
Paclitaxel is an effective agent in the treatment of breast, ovarian,
lung, and head and neck cancers (8). Paclitaxel promotes tubulin
polymerization, thus altering the dynamic equilibrium of assembling and
disassembling of microtubules and causing mitotic arrest of dividing
cells (9). However, the precise mechanisms of paclitaxel-induced
cytotoxicity and apoptosis have not been elucidated. Recently, several
studies have suggested that paclitaxel-induced apoptosis may be
mediated by loss of Bcl-2 function as a result of phosphorylation by
activated Raf-1 kinase (10-12). Other studies, however, have not
confirmed this observation. Ibrado et al. (13) reported that
paclitaxel-induced apoptosis in human myeloid leukemia HL-60 cells was
not associated with activation of Raf-1 kinase and Bcl-2
phosphorylation (13). These controversial findings prompted us to study
more in detail the relationship between paclitaxel-induced Bcl-2
phosphorylation and apoptosis in HeLa cervical carcinoma cells and
SKOV3 ovarian carcinoma cells. The results presented here indicate that
hyperphosphorylation of Bcl-2 is temporally associated with
paclitaxel-induced M phase arrest but not with apoptosis. Results with
HeLa cells synchronized by either nocodazole or double thymidine
blockade confirmed that the phosphorylation of Bcl-2 is a marker of M
phase events and failed to demonstrate a direct linkage between Bcl-2
phosphorylation and induction of apoptosis. Additional studies with
inhibitors of phosphatase and kinase in nocodazole-blocked M phase
cells further confirmed that the phosphorylation of Bcl-2 is tightly
linked to the regulation of M phase events.
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MATERIALS AND METHODS |
Chemicals and Antibodies--
Paclitaxel, nocodazole, and
thymidine were purchased from Sigma. Okadaic acid, staurosporine,
genistein, and histone H1 were purchased from Boehringer Mannheim. Cell
culture medium was obtained from Life Technologies, Inc. Monoclonal
anti-Bcl-2 (Ab-1) antibody, anti-Cdc2 (Ab-1), and anti-cyclin B1 (Ab-2)
were purchased from Calbiochem. Polyclonal anti-Raf-1 antibody was
purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Cell Culture and Synchronization--
HeLa and SKOV3 cells were
obtained from American Type Culture Collection (Rockville, MD) and
grown in Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum and 250 mM L-glutamine.
Cells were maintained at 37 °C in a humidified atmosphere of 5%
CO2, 95% air incubator. Exponentially growing cells were
exposed to paclitaxel at the indicated concentrations for different
lengths of time. Tissue culture samples were taken at the indicated
times, and cell lysates were prepared for Western blot analysis or for
kinase assay. For cell synchronization at metaphase, HeLa cells (5 × 106 cells/25-cm2 flask) were exposed to 100 ng/ml nocodazole at 37 °C for 16 h. After treatment, metaphase
cells were collected by the gentle shakeoff method and washed three
times with fresh medium. For relieving cells from M phase arrest, cells
(1 × 106) were replated in a 60 × 15-mm dish
and incubated at 37 °C in fresh medium for different times. For
G1/S phase synchronization, cells (1 × 106 cells in 60 × 15-mm dishes) were treated with
thymidine (two 16-h periods of exposure to 2 mM thymidine
separated by a 10-h exposure without thymidine) as described previously
(14). At the indicated time points after release, cells were washed
once with cold Ca2+, Mg2+-free
phosphate-buffered saline and stored at
90 °C until use.
Immunobloting--
Cells were lysed with lysis buffer containing
50 mM Tris-HCl (pH 7.4), 0.1% Triton X-100, 1% SDS, 250 mM NaCl, 15 mM MgCl2, 1 mM dithiothreitol, 2 mM EDTA, 2 mM
EGTA, 25 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. The protein amount in each sample was determined by a DC protein assay kit (Bio-Rad). Equal amounts of lysate were subjected to electrophoresis in
a 0.1% SDS, 10% polyacrylamide gel. The proteins were transferred onto nitrocellulose membrane. After blocking with 5% nonfat milk in
Tris-buffered saline-Tween buffer at room temperature for 1 h,
Bcl-2, Cdc2, and cyclin B1 were probed with monoclonal anti-Bcl-2, anti-Cdc2, or anti-cyclin B1 antibodies. The immunoblots were analyzed
using an ECL detection system according to the manufacturer's recommendation (Amersham Pharmacia Biotech). The relative amounts of
Cdc2, cyclin B1, and phosphorylated and unphosphorylated Bcl-2 protein
were quantitatively measured by laser scanning densitometry (Molecular
Dynamics, Sunnyvale, CA).
Immunoprecipitation and Kinase Assay--
Cells (1 × 106 cells) were aliquoted from the cultures at the
indicated time points and solubilized in 0.5 ml of lysis buffer containing 50 mM Tris-HCl (pH 7.4), 250 mM
NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1% Triton X-100. After incubation at 4 °C for 15 min, the lysate was separated by
centrifugation at 4,000 rpm for 10 min at 4 °C. The protein content
in each sample was determined as described above using a DC protein
assay kit and adjusted accordingly before the assay. Lysate (0.5 ml)
was incubated with 5 µg monoclonal anti-Cdc2 and anti-cyclin B1
antibodies and 50 µl of protein A/protein G conjugated agarose
(Calbiochem) at 4 °C overnight. After washing three times with lysis
buffer and once with reaction buffer, the immunoprecipitate complex was collected and incubated at 30 °C in 50 µl of kinase reaction
mixture containing 50 mM Tris-HCl (pH, 7.4), 10 mM MgCl2, 1 mM dithiothreitol, 10 µM ATP, 5 µCi of [
-32P]ATP, and 0.5 mg/ml of histone H1 for 15 min. The reaction was terminated by the
addition of 20 µl of 4× Laemmli's sample buffer and boiling for 5 min. The 32P-phosphorylated histone H1 was separated by
0.1% SDS, 10% polyacrylamide gel, and determined either by
autoradiography using Kodak X-Omat film or by liquid scintillation
counting.
To assay Bcl-2 phosphorylation by Cdc2/cyclin B1 kinase, Bcl-2 and
Cdc2/cyclin B1 were immunoprecipitated from 1 × 107
HeLa cells by monoclonal anti-Bcl-2 and anti-Cdc2/anti-cyclin B1
antibodies as described above. The reaction of Bcl-2 phosphorylation was carried out in 50 µl of reaction mixture containing 50 mM Tris-HCl (pH, 7.4), 10 mM MgCl2,
1 mM dithiothreitol, 10 µM ATP, 5 µCi of
[
-32P]ATP, and Bcl-2 and Cdc2/cyclin B1 immune
complex. After incubation at 30 °C for 15 min, the reaction was
terminated by addition of 4× Laemmli's sample buffer. After boiling
for 5 min, the samples were subjected to a 10% SDS-polyacrylamide gel
electrophoresis. Proteins were transferred to the nitrocellulose
membrane and probed with monoclonal anti-Bcl-2 antibody with ECL
detection as described above. The 32P-incorporated Bcl-2
protein was analyzed by autoradiography.
Mitosis and Apoptosis Assay--
The number of mitotic cells was
determined in cells stained with Wright-Giemsa dye solution. Apoptotic
cells were detected using a
Tunel1 reaction kit according
to the manufacturer's recommendation (Boehringer Mannheim). After the
reaction, the fluorescence-labeled cells were determined by flow
cytometry (Epics Profile Analyzer, Coulter Co., Miami, FL). For
determination of paclitaxel-induced DNA fragmentation, cells were
solubilized with 0.5 ml of lysis buffer containing 10 mM
Tris-HCl (pH, 8.0), 200 mM NaCl, and 0.2% Triton X-100 at room temperature for 30 min. After centrifugation at 14,000 rpm for 5 min, the supernatant fraction was collected and fragmented DNA was
precipitated with 100 mM NaCl and equal volume isopropanol at
20 °C overnight. DNA was dissolved in Tris-EDTA buffer
containing 20 units of RNase and incubated at 60 °C for 60 min.
After electrophoresis in 1% agarose gel, DNA was stained with ethidium
bromide, and resulting DNA fragmentation was visualized by UV
illumination (15).
 |
RESULTS |
Effect of Paclitaxel on Mitotic Arrest, Cyclin B1 Accumulation,
Activation of Cdc2/Cyclin B1 Kinase, Apoptosis, and Phosphorylation of
Bcl-2 Protein--
Initially, we treated HeLa cells continuously with
50 ng/ml paclitaxel (a concentration that corresponds to the
ID90) and determined drug-induced M phase arrest by using
Wright-Giemsa dye staining. About 2% cells were in M phase at
baseline. Paclitaxel treatment resulted in a steady increase of cells
arrested at M phase which was maximum at 24 h and decreased
thereafter, i.e. 82% in M phase at 24 h and barely no
cells in M phase at 48-60 h. Because M phase is associated with
accumulation of B type cyclins and activation of Cdc2/cyclin B complex
kinase (16, 17), we determined the effect of paclitaxel on the levels
of Cdc2 and cyclin B1 and activity of Cdc2/cyclin B1 kinase. Similar to
M phase arrest, paclitaxel treatment caused a gradual increase in cyclin B1 amounts that peaked at 24 h and markedly decreased at 48-60 h. Under the same experimental conditions, the Cdc2 levels were
relatively constant. As expected, the time-course of drug-induced stimulation of Cdc2/cyclin B1 kinase activity was similar to those of
drug-induced M phase arrest and induction of cyclin B1 (Fig. 1). In summary, paclitaxel-induced M
phase arrest, accumulation of cyclin B1, and stimulation of Cdc2/cyclin
B1 kinase exhibited a bell shape time-course with the peak of the curve
at 24 h.

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Fig. 1.
Effect of paclitaxel on Cdc2 and cyclin B1
expression and Cdc2/cyclin B1 kinase activity in HeLa cells. Cells
were continuously exposed to 50 ng/ml paclitaxel. Equal amounts of
lysate (50 µg of protein) from each sample were subjected to 10%
SDS-polyacrylamide gel electrophoresis; Cdc2 and cyclin B1 were
determined by Western blot analysis with ECL detection. For Cdc2/cyclin
B1 kinase assay, the immune complex was immunoprecipitated by anti-Cdc2
and anti-cyclin B1 antibodies, and kinase activity was determined by
[ -32P]ATP incorporation into histone H1, as described
under "Materials and Methods." A, Cdc2 and cyclin B1
levels determined by Western blot analysis; B, relative
levels of Cdc2 and cyclin B1 determined by a laser scanning
densitometer; C, 32P incorporation into histone
H1 determined by autoradiography; D, relative Cdc2/cyclin B1
kinase determined by 32P incorporation in histone H1.
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Next, we determined the effect of paclitaxel in inducing apoptosis in
HeLa cells. Drug treatment was the same as described above. Aliquots of
cells were taken at different time points and apoptotic cells
determined by morphology and Tunel reaction assay. As shown in Fig.
2, A and B, no
apoptotic cells were observed at 6 h; about 8-18% of cells were
apoptotic by 12-24 h. The number of apoptotic cells markedly increased
after 24 h, with 60-80% apoptotic cells at 36-48 h and 96% at
60 h. To confirm that drug-induced cell death was due to
apoptosis, DNA obtained from cells at different time points was
subjected to a 1% agarose gel electrophoresis, stained with ethidium
bromide, and visualized by UV illumination. As shown in Fig.
2C, a typical apoptotic hallmark, cleavage of genomic DNA
into multiple internucleosome (~200 base pairs), appeared at 24 h, and the extent of DNA fragmentation increased with time. These data
further confirm our previous findings in KB and SKOV3 cells that
paclitaxel-induced mitotic arrest precedes apoptosis (18), and are
consistent with the report by Jordan et al. (19).

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Fig. 2.
Paclitaxel-induced apoptosis in HeLa
cells. Cells were continuously exposed to 50 ng/ml paclitaxel for
different times. Apoptotic cells were determined by observation of
morphological changes, Tunel reaction, and DNA fragmentation assay as
described under "Materials and Methods." A, apoptotic
cells induced by paclitaxel for different times and determined by Tunel
reaction assay; B, apoptotic cells determined by microscopic
observation ( ------ ) or Tunel assay ( ------ ); C,
DNA fragmentation determined by 1% agarose gel electrophoresis.
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Finally, we determined whether paclitaxel-induced apoptosis is
associated with Bcl-2 phosphorylation. We treated cells continuously with 50 ng/ml paclitaxel, collected cell aliquots from culture at the
indicated time-points, and lysed cells with lysis buffer. An equal
amount of lysate (50 µg protein) from each sample was subjected to a
10% SDS-polyacrylamide gel electrophoresis, and Bcl-2 protein was
probed by monoclonal anti-Bcl-2 antibody with ECL detection. The
results presented in Fig. 3A,
a typical and reproducible Western blot analysis, revealed that
paclitaxel treatment for 12 h clearly altered the Bcl-2
electrophoretical mobility pattern, resulting in two bands, one
corresponding to the 26 kDa unphosphorylated Bcl-2 and another to the
slower mobility phosphorylated Bcl-2 protein. At 24 h, two slower
Bcl-2 bands appeared, whereas the 26 kDa Bcl-2 band had almost
completely disappeared, indicating that most Bcl-2 protein had been
subjected to phosphorylation and hyperphosphorylation. At 36 h,
the 26 kDa of Bcl-2 again became predominant, but two slower bands
still could be seen. However, at 48-60 h, the slower bands
corresponding to the hyperphosphorylated and phosphorylated Bcl-2 had
completely disappeared, and only the 26-kDa Bcl-2 protein band could be
seen. We used a laser scanning densitometer to quantitatively measure
the unphosphorylated and phosphorylated Bcl-2 protein bands and
found that paclitaxel-induced Bcl-2 phosphorylation exhibited a good
correlation with drug-induced M phase arrest (Fig. 3B).
Taken together, as presented in Fig. 4,
we conclude that paclitaxel-induced Bcl-2 phosphorylation is associated
in time with M phase arrest and activation of Cdc2/cyclin B kinase,
thus preceding apoptosis, and that at the time of massive apoptosis the
Bcl-2 protein is fully unphosphorylated (Fig. 4).

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Fig. 3.
Paclitaxel-induced Bcl-2 phosphorylation and
M phase arrest in HeLa cells. Cells were continuously exposed to
50 ng/ml paclitaxel. Mitotic cells were determined by counting
Wright-Giemsa-stained cells and Bcl-2 phosphorylation was determined by
Western blot analysis with ECL detection as described under
"Materials and Methods." A, Bcl-2 phosphorylation
induced by paclitaxel after exposure for different times. Bands
representing hyperphosphorylated (U), phosphorylated
(M), and unphosphorylated Bcl-2 protein (B) were
detected with ECL reaction; B, the correlation between M
phase arrest and Bcl-2 phosphorylation after exposure to
paclitaxel.
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Fig. 4.
Time course studies of paclitaxel-induced M
phase arrest, Cdc2/cyclin B1 kinase activity, Bcl-2 protein
phosphorylation, and apoptosis in HeLa cells. Cells were
continuously exposed to 50 ng/ml paclitaxel. Drug-induced M phase
arrest, Cdc2/cyclin B1 kinase, Bcl-2 phosphorylation, and apoptosis
were determined as described under "Materials and Methods."
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To confirm that paclitaxel-induced Bcl-2 phosphorylation is associated
with mitosis in a different cell line, we treated human ovarian
carcinoma SKOV3 cells continuously with 50 ng/ml paclitaxel and
determined Bcl-2 phosphorylation, mitosis and apoptosis at different
time points. As shown in Fig. 5, the
patterns of paclitaxel-induced Bcl-2 phosphorylation, M phase arrest
and apoptosis in SKOV3 cells are similar to those in HeLa cells
described above. Thus it indicates that the association of drug-induced
Bcl-2 phosphorylation with M phase arrest, not with apoptosis is not
restricted to HeLa cells.

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Fig. 5.
Paclitaxel-induced Bcl-2 phosphorylation, M
phase arrest, and apoptosis in SKOV3 cells. Cells were
continuously exposed to 50 ng/ml paclitaxel. The number of mitotic
cells was determined by counting Wright-Giemsa-stained cells, apoptosis
was determined by morphological observation and Bcl-2 phosphorylation
was determined by Western blot analysis with ECL detection as described
under "Materials and Methods." A, Bcl-2 phosphorylation
induced by paclitaxel after exposure for different times. Bands
representing hyperphosphorylated (U), phosphorylated
(M), and unphosphorylated Bcl-2 protein (B) were
detected with ECL reaction; B, time curves of mitotic and
apoptotic cells and Bcl-2 phosphorylation after exposure to
paclitaxel.
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Bcl-2 Phosphorylation in Synchronized HeLa Cells--
To further
confirm that Bcl-2 protein phosphorylation is associated with M phase
arrest, we used synchronized HeLa cells to determine Bcl-2
phosphorylation at different phases of the cell cycle. First, we
treated cells with 100 ng/ml nocodazole for 16 h and collected M
phase blocked cells as described in Materials and Methods. After
washing three times with fresh medium, cells were replated and
incubated in fresh medium. The number of mitotic cells and the amount
of phosphorylated Bcl-2 protein were determined at different
time-points after release as described above. As shown in Fig.
6A, about 91% of cells were
in M phase at time 0 and about 86% at 1 h post-release. The
number of mitotic cells decreased to 44% at 2 h post-release and
to 3-7% cells at 3-5 h, indicating that most cells had been relieved
from nocodazole-blocked M phase and had progressed into interphase by
3 h of release. Western blot analysis revealed that, from time 0 to 1 h after release, the Bcl-2 protein exhibited two slower bands
corresponding to hyperphosphorylated and phosphorylated protein, with
no dephosphorylated protein band. In contrast, 2 h after release,
when 56% of cells had exited M phase, about 50% of Bcl-2 protein was
dephosphorylated. After 3-5 h of release, almost all cells were in
interphase and only a 26-kDa dephosphorylated band was displayed in the
gel. We also determined the apoptotic cells at different time points after release and only found a few apoptotic cells (<10%) up to 48 h after release.

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Fig. 6.
A Bcl-2 phosphorylation in M phase cells
blocked by nocodazole or paclitaxel. HeLa cells were exposed to
either 100 ng/ml nocodazole or 50 ng/ml paclitaxel for 16 h. M
phase blocked cells were harvested by shake off, washed three times
with fresh medium and reincubated in fresh medium. At different times
after post-incubation, the number of mitotic cells was determined by
counting Wright-Giemsa-stained cells, apoptosis was determined by
morphological changes, phosphorylation of Bcl-2 was determined by
Western blot analysis as described under "Materials and Methods."
A, time course study of M phase arrest, apoptosis induction,
and Bcl-2 phosphorylation in nocodazole-blocked M phase cells at the
indicated times post-incubation. B, time course study of M
phase arrest, apoptosis induction, and Bcl-2 phosphorylation in
paclitaxel-blocked M phase cells at the indicated times
post-incubation.
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To compare side by side the effects of nocodazole and paclitaxel under
the same experimental conditions, we treated HeLa cells with 50 ng/ml
paclitaxel for 16 h and collected the M phase cells as described
above. Cell aliquots were collected at different times after drug
incubation. The number of mitotic and apoptotic cells and the extent of
Bcl-2 phosphorylation were determined as described above. As shown in
Fig. 6B, the M phase blockade caused by paclitaxel was very
slowly reversible. For example, about 90% cells were in mitosis during
the first 6 h post-incubation, decreasing to 80-60% at 12-24 h
post-incubation. Again, the extent of Bcl-2 phosphorylation was tightly
associated with the number of mitotic cells, i.e. about 90%
of Bcl-2 protein was phosphorylated during the first 6 h
post-incubation, decreasing to 80-50% at 12-24 h. In contrast, only
3-5% cells were in mitosis and there was no detectable phosphorylated
Bcl-2 24 h after a 16 h exposure to nocodazole. In addition,
the persistence of mitotic arrest and phosphorylated Bcl-2 in
paclitaxel-treated cells was associated with massive (>80%) apoptosis
at 48 h post-incubation versus <5% after
nocodazole.
Finally, because a 16 h exposure to nocodazole only caused a
rapidly and completely reversible M phase arrest and minimal apoptosis,
we investigated whether a continuous exposure to 100 ng/ml nocodazole
could induce apoptosis in HeLa cells. As shown in Fig.
7, continuous exposure to nocodazole
caused a more prolonged M phase arrest and Bcl-2 phosphorylation than a
16 h exposure, and resulted in massive (>80%) apoptosis at
60 h, with a pattern very similar to that observed in cells
continuously exposed to paclitaxel (Fig. 4). Altogether, these results
confirm a tight temporal association between Bcl-2 phosphorylation and
M phase arrest, and indicate that with either agent apoptosis is only induced under experimental conditions that result in prolonged mitotic
arrest and Bcl-2 phosphorylation.

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Fig. 7.
Bcl-2 phosphorylation, M phase arrest and
apoptosis induced by continuous exposure to nocodazole in HeLa
cells. Cells were continuously exposed to 100 ng/ml nocodazole at
37 °C for the indicated periods of time. Mitotic cells were
determined by counting Wright-Giemsa stained cells, apoptosis was
determined by morphological changes and Bcl-2 phosphorylation was
determined by Western blot analysis with ECL detection as described
under "Materials and Methods." A, Bcl-2 phosphorylation
induced by continuous exposure to nocodazole for different times. Bands
representing hyperphosphorylated (U), phosphorylated
(M), and unphosphorylated Bcl-2 protein (B) were
detected with ECL reaction; B, time curves of mitotic and
apoptotic cells and Bcl-2 phosphorylation after exposure to
nocodazole.
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Next, we used double thymidine to synchronize HeLa cells and determined
the presence of phosphorylated Bcl-2 at different phases of the cell
cycle. As shown in Fig. 8, about 70% of
cells were synchronized at G1 phase after double thymidine
block (two periods of exposure with a 10-h interval of incubation in
normal medium). Accumulation of cells at G2/M was started
to be observed at 6 h post-block release, peaked (about 60%
G2/M cells) at 10 h, and still remained significant
(about 40% G2/M cells) at 12 h. Interestingly,
Western blot analysis revealed that the slower mobility band,
representing phosphorylated Bcl-2 protein, appeared at 6 h after
release and lasted until 12 h after release. The phosphorylated
band disappeared at 24 h, when 80% of cells were in
G1 phase and only 8% of cells in G2/M phase.
These results further confirm that Bcl-2 protein phosphorylation is
associated with cells at G2/M phase.

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Fig. 8.
Bcl-2 phosphorylation in HeLa cells
synchronized by double thymidine block. Cells were synchronized
with double thymidine block as described under "Materials and
Methods." At different times post-release, cell aliquots were taken,
the cell cycle distribution was determined by flow cytometry, and the
Bcl-2 protein phosphorylation was determined by Western blot analysis
as described under "Materials and Methods." A, cell
cycle distribution determined by flow cytometry; B, changes
in Bcl-2 protein phosphorylation at different times post-release, bands
representing phosphorylated (U) and unphosphorylated
(B) Bcl-2 detected with ECL reaction.
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Effects of Phosphatase Inhibitor and Protein Kinase Inhibitor on
Bcl-2 Phosphorylation and Mitosis--
Although the function of
phosphorylated Bcl-2 and the sites of phosphorylation on the Bcl-2
protein remain to be defined, it has been speculated that
serine/threonine kinases and phosphatases may be involved in the
regulation of phosphorylation and dephosphorylation of Bcl-2 protein
(20). To further study the association between Bcl-2 phosphorylation
and mitosis, we used inhibitors of phosphatase and protein kinase to
examine whether the alteration in Bcl-2 phosphorylation by these
inhibitors could alter the exit of cells from mitosis. First, we
synchronized HeLa cells in M phase with nocodazole and then replated
the M phase cells in fresh medium in the absence or presence of 1 µM okadaic acid, a phosphatase inhibitor. After
incubation for the indicated periods of time, cell aliquots were taken
from the culture, and the number of mitotic cells and extent of Bcl-2
phosphorylation were determined as described above. As shown in Fig.
9A, treatment with okadaic
acid prevented or delayed the exit of the cells from mitosis and the
dephosphorylation of phosphorylated Bcl-2. For example, 69% cells
remained in M phase and the hyperphosphorylated or phosphorylated Bcl-2
bands were still predominant at 4-6 h post-release in the presence of okadaic acid, versus only 4-9% cells and barely no
phosphorylated Bcl-2 bands in the absence of inhibitor. Next, we used
staurosporine, a serine/threonine kinase inhibitor, to examine whether
the inhibition of Bcl-2 phosphorylation could facilitate the exit of
cells from mitosis and entry into interphase. Cells arrested at M phase
after exposure to 100 ng/ml nocodazole for 16 h were used for
these experiments. As shown in Fig. 9B, treatment of cells
arrested at M phase by nocodazole with 100 nM staurosporine
for 60 min resulted in a massive entry of mitotic cells into
interphase, whereas 80% cells remained in M phase in the absence of
staurosporine. As expected, the presence of staurosporine for 60 min
resulted in complete dephosphorylation of the Bcl-2 protein, whereas
the Bcl-2 protein remained at the hyperphosphorylation state in the absence of inhibitor. Finally, we used genistein, a tyrosine kinase inhibitor, to determine the effect of this inhibitor on cell entry into
interphase and Bcl-2 dephosphorylation. Genistein did not alter the
number of mitotic cells nor the extent of Bcl-2 phosphorylation in
cells treated with 25 µM genistein for 90 min after
release (Fig. 9C). These findings suggest that
serine/threonine kinases, but not tyrosine kinases, are involved in the
regulation of Bcl-2 phosphorylation and M phase transition. These
results are consistent with those reported by Chen and Faller (21).

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Fig. 9.
Effect of okadaic acid, staurosporine, and
genistein on exit from M phase into interphase and Bcl-2
phosphorylation in HeLa cells. Cells were blocked in M phase by
exposure to 100 ng/ml nocodazole for 16 h and then incubated in
fresh medium with or without 1 µM okadaic acid, 100 nM staurosporine, or 25 µM genistein for the
indicated periods of time. At different times post-release, the number
of mitotic cells was determined by counting chromatin condensation in
Wright-Giemsa stained cells. Phosphorylation of Bcl-2 protein was
determined by Western blot analysis as described under "Materials and
Methods." A, okadaic acid prevents dephosphorylation of
Bcl-2 and exit from mitosis into interphase; B,
staurosporine stimulates dephosphorylation of Bcl-2 and enhances exit
from mitosis into interphase; C, genistein does not affect
phosphorylation of Bcl-2 nor M phase blockade, I, Bcl-2
protein from interphase cells indicating unphosphorylated state.
|
|
Role of Cdc2/Cyclin B Kinase in Bcl-2 Phosphorylation--
Because
Bcl-2 phosphorylation is temporally associated with activation of
Cdc2/cyclin B1 kinase, we investigated the potential role of
Cdc2/cyclin B1 complex kinase in phosphorylating Bcl-2. To determine
whether Bcl-2 phosphorylation may be associated with activation of
Cdc2/cyclin B1 kinase, we used monoclonal anti-Bcl-2 antibody to
immunoprecipitate Bcl-2 protein from HeLa cells. Equal amounts of Bcl-2
detected with ECL were added to a reaction mixture consisting of 50 mM Tris-HCl, 10 mM MgCl2, 10 µM ATP, 5 µCi of [
-32P]ATP and
Cdc2/cyclin B1 complex immunoprecipitated from untreated and
paclitaxel-treated cells. The paclitaxel-induced stimulation of
Cdc2/cyclin B1 kinase activity was determined by using histone H1 as a
substrate as described above. As shown in Fig.
10, the Bcl-2 protein
immunoprecipitated from untreated cells was markedly phosphorylated by
the Cdc2/cyclin B1 kinase complex precipitated from paclitaxel-treated
cells, although the extent of such phosphorylation was lower than that
using histone H1 as a substrate. In contrast, no Bcl-2 protein was
phosphorylated by the Cdc2/cyclin B1 kinase complex precipitated from
untreated cells. Under the same experimental conditions, other cell
cycle related kinases precipitated from paclitaxel-treated cells, such
as Cdc2/cyclin A and Cdk2/cyclin E, failed to phosphorylate Bcl-2
protein, indicating that phosphorylation of Bcl-2 may be at least in
part associated with the activation of M phase-related kinase (data not
shown).

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|
Fig. 10.
Phosphorylation of Bcl-2 protein in a
cell-free system. HeLa cells were treated with 50 ng/ml paclitaxel
or the same volume of medium as control for 24 h. At the
completion of drug incubation, the Cdc2/cyclin B1 complex was
immunoprecipitated from paclitaxel-treated and control cells with
monoclonal anti-Cdc2 and anti-cyclin B1 antibodies, and the Bcl-2
protein was immunoprecipitated from untreated cells with monoclonal
anti-Bcl-2 antibody. Cdc2/cyclin B1 kinase activity was determined by
32P incorporation into histone H1 as a substrate and
32P incorporation into Bcl-2 as described under
"Materials and Methods." A, Cdc2/cyclin B1 kinase
activity determined by 32P incorporation into histone H1 in
control and paclitaxel-treated cells; B, Bcl-2 protein
prepared from immunoprecipitation in interphase cells and detected with
ECL reaction, 1 and 2 indicating the similar
amount of Bcl-2 for phosphorylation assay by Cdc2/cyclin B1 complex;
C, autoradiography of 32P incorporation into
Bcl-2 by Cdc2/cyclin B1 immunoprecipitated from control or
paclitaxel-treated cells; M, marker of prestained protein
molecular weight.
|
|
 |
DISCUSSION |
The results of the present study demonstrate that the anti-tubulin
agents paclitaxel and nocodazole induce transient Bcl-2 phosphorylation
in close association with mitotic arrest and cyclin B1 kinase
activation. Bcl-2 phosphorylation of variable duration was observed in
HeLa cells synchronized with double thymidine when they reach
G2/M phase, HeLa cells transiently arrested in mitosis by
nocodazole, and HeLa cells arrested in mitosis prior to apoptosis after
continuous exposure to paclitaxel and nocodazole. The duration of
Bcl-2 phosphorylation was about 3-6 h in cells synchronized with
double thymidine or transiently arrested in mitosis after a 16-h
exposure to nocodazole. In both cases, no apoptosis was observed. In
contrast, Bcl-2 phosphorylation lasted >24 h in cells continuously
exposed to paclitaxel and nocodazole and in these situations massive
apoptosis was observed 24-36 h after maximum mitotic arrest. However,
at the time of massive apoptosis all Bcl-2 was dephosphorylated.
Altogether, these results do not appear to support the recently stated
hypothesis that inactivation of Bcl-2 by phosphorylation is a major
potential mechanism of paclitaxel-induced apoptosis (4, 7, 11, 12). If
Bcl-2 phosphorylation was a major determinant of paclitaxel-induced apoptosis, one would have expected to observe a similar apoptotic effect in nocodazole-synchronized mitotic cells in which >90% of
Bcl-2 protein was phosphorylated. However, we can not rule out nor
prove from our studies that it is not Bcl-2 phosphorylation per se but
a persistent (>24 h) Bcl-2 inactivation through phosphorylation the
important determinant of paclitaxel-induced apoptosis.
Another possible explanation for the discrepant conclusions between
ours and previous studies may be related to the time frame of the
experiments, 24 h in previous studies as opposed to 48-60 h in
our study. If our studies had not included determinations at 48-60 h,
we would have not been able to demonstrate that paclitaxel-induced Bcl-2 phosphorylation peaks at 24 h, as well as other markers of
mitotic arrest such as accumulation of cyclin B1 and activation of
Cdc2/cyclin B1 kinase, and that all the mitotic markers are completely
reversed by 48-60 h, when massive apoptosis is paramount (Fig. 3).
Interestingly, our results are consistent with the recent report by
Ibrado et al. (13) in HL-60 human leukemia cells
demonstrating complete dephosphorylation of Bcl-2 at the time of
paclitaxel-induced DNA degradation.
The fact that Bcl-2 is dephosphorylated at the time of massive
apoptosis does not rule out in principle the possibility that a
preceding Bcl-2 phosphorylation lasting more than a certain critical
period of time might be sufficient to trigger the apoptotic pathway. However, the proposed mechanism of loss of Bcl-2 antiapoptotic activity appears to be mediated by a decreased ability of the phosphorylated protein to interact with proapoptotic proteins such as
bax, in which case one would expect a persistently phosphorylated and
non-functional Bcl-2 protein at the time of paclitaxel-induced massive apoptosis (22, 23).
The mechanism of Bcl-2 phosphorylation in mitotic cells has not been
fully elucidated. There is evidence that Raf-1 can phosphorylate the
Bcl-2 protein in the outer mitochondrial membrane (24, 25). Blagosklonny et al. (10, 11) reported that
paclitaxel-induced apoptosis was associated with the activation of
Raf-1 kinase and phosphorylation of Bcl-2 in MCF-7 cells. However, some
investigators have shown that depletion of Raf-1 kinase activity by the
specific inhibitor geldanamycin did not affect paclitaxel-induced Bcl-2 phosphorylation and apoptosis (13). To further investigate whether Raf-1 is responsible for phosphorylating Bcl-2 in mitotic cells, we
used the Jurkat cells and the mutant cells J.CaM.1, which are deficient
in Lck gene and lack Raf-1 kinase activation in M phase (26). Our preliminary results indicate that the pattern of Bcl-2 phosphorylation induced by paclitaxel in Jurkat cells is similar to
that in J.CaM.1 cells, thus suggesting that Raf-1 kinase may not be
involved in paclitaxel-induced Bcl-2 phosphorylation (data not shown).
Another possibility would be that Bcl-2 is phosphorylated by
Cdc2/cyclin B1 kinase since Cdc2/cyclin B1 kinase activation and Bcl-2
phosphorylation occur at the same time. It is well known that several
proteins are phosphorylated in M phase by Cdc2/cyclin B kinase, and
that these phosphorylated proteins play crucial roles in regulating the
mitotic process, including chromatin condensation, nuclear envelope
breakdown, and formation of the mitotic spindle. We were able to
demonstrate using immune complex kinase assays in cell free systems
that in fact the Cdc2/cyclin B1 complex can phosphorylate Bcl-2,
although the extent of phosphorylation was lower than with histone H1
as a substrate (Fig. 10). It remains to be determined whether this is
also the case in whole cell systems since the Cdc2/cyclin B1 complex is
located in the nucleus and the Bcl-2 protein in the mitochondrial
membrane. However, in favor of the possibility that Bcl-2 may be
phosphorylated by Cdc2/cyclin B1 kinase is the observation by Lu
et al. that the subcellular distribution of Bcl-2 protein
determined by either immunocytochemistry or immunoelectron microscopy
was dependent on cell cycle, i.e. Bcl-2 protein was
localized in the cytoplasm at interphase and in the nucleus, in
particular in chromosomes, at M phase (27).
Our results indicate that a phosphorylated Bcl-2 protein is a landmark
of mitotic cells. Like in the case of M phase-related cyclins and
kinases, it is reasonable to assume that Bcl-2 phosphorylation and
dephosphorylation may play an important role in regulating M phase
specific events, such as facilitation of mitotic-related kinase into
nuclei, breaking down of nuclear envelope, condensation of chromatin,
formation of the mitotic spindle, or chromosome segregation, and
determining the progression of cells from G2 into mitosis
and from mitosis into interphase, respectively. The elucidation of the
mechanisms by which Bcl-2 phosphorylation and dephosphorylation affect
cell cycle progression deserves, therefore, further investigation. In
our studies, Bcl-2 phosphorylation was found to be regulated by
unidentified phosphatases and serine/threonine kinase since treatment
with either okadaic acid or staurosporine strikingly altered Bcl-2
phosphorylation and delayed or enhanced, respectively, exit from
mitosis and entry into interphase. The observation that kinase and
phosphatase inhibitors may disrupt the equilibrium between Bcl-2
phosphorylation and dephosphorylation and impair the normal progression
of the cell cycle in mitosis could provide a therapeutic opportunity
and be exploited to potentiate the effects of paclitaxel or other
chemotherapeutic agents.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grant CA50270.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.
¶
To whom requests for reprints should be addressed: Dept. of
Thoracic/Head and Neck Medical Oncology, Box 080, The University of
Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: 713-792-6363; Fax: 713-796-8655.
1
The abbreviation used is: Tunel, terminal
deoxynucleotidyl transferase-mediated dUTP nick end-labeling.
 |
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