From the Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan
Received for publication, November 25, 2002, and in revised form, February 14, 2003
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ABSTRACT |
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kpm is a human serine/threonine
kinase that is homologous to Drosophila tumor suppressor
warts/lats and its mammalian homologue LATS1. In order to define
the biological function of kpm, we generated stable transfectants of
wild-type kpm (kpm-wt), a kinase-dead mutant of
kpm (kpm-kd), and luciferase in HeLa Tet-Off
cells under the tetracycline-responsive promoter. Western blot analysis
showed that high levels of expression of kpm-wt as well as kpm-kd with an apparent mass of 150 kDa were induced after the removal of doxycycline. Induction of kpm-wt expression resulted in a marked decline in viable cell number measured by both trypan blue dye exclusion and MTT assay, whereas that of kpm-kd or luciferase had no
effect. We then analyzed the cell cycle progression and apoptosis upon
induction of kpm expression. 2-3 days after removal of doxycycline,
cells underwent G2/M arrest, demonstrated by flow cytometric analysis of propidium iodide incorporation and MPM-2 reactivity. In vitro kinase assay showed that induction of
kpm-wt led to down-regulation of kinase activity of the
Cdc2-cyclin B complex, which was accompanied by an increase in the
hyperphosphorylated form of Cdc2 and a change of phosphorylation status
of Cdc25C. Furthermore, both DAPI staining and TUNEL assay
showed that the proportion of apoptotic cells increased as kpm
expression was induced. Taken together, these results indicate that kpm
negatively regulates cell growth by inducing G2/M arrest
and apoptotic cell death through its kinase activity.
kpm (1) is a novel protein kinase that was molecularly cloned from
a human myeloid precursor cell line KG-1a (2) by degenerate PCR
targeted for the conserved serine/threonine kinase domain. The
sequencing and homology search revealed that kpm is homologous to a Drosophila melangaster gene,
warts, or alternatively lats (3, 4).
warts/lats was originally identified by mitotic recombination of somatic cells and screening for homozygous mutants with overproliferation phenotype. Somatic cells mutated for this gene
undergo extensive proliferation and form large tumors with abnormal
morphologies, suggesting that warts/lats functions as a tumor
suppressor. It is now recognized that warts/lats belongs to a subfamily
of protein kinases with a common characteristic structure in the kinase
domain consisting of dbf2 of budding yeast (5), orb6 of fission
yeast (6), cot-1 of Neurospora crassa (7), ndr of various
species (8), myotonic dystrophy protein kinase (DMPK) of human (9),
Rho-associated kinases of mammalian (10), and some other related
kinases. It is noted that all the members of this family have been
shown to be involved in the regulation of cell cycle progression.
A mammalian homologue of warts/lats, named
LATS1 (11, 12), has been isolated and extensively studied.
Introduction of human LATS1 into Drosophila
warts/lats mutants could prevent tumor formation and support normal
development, demonstrating that the functions of these genes are
conserved from flies to humans. Moreover, mice deficient for mouse
LATS1 developed soft tissue sarcomas and ovarian tumor (13),
indicating that this gene functions as a tumor suppressor also in
mammals. In terms of cell cycle regulation, LATS1 protein has been
shown to be phosphorylated and bind to Cdc2 at early mitosis (11).
These data suggest that LATS1 is an authentic homologue of
Drosophila warts/lats and has crucial functions in
regulation of cell growth.
kpm is distinct from LATS1 but these two genes
are highly homologous to each other especially at the kinase domain. A
cDNA identical to kpm was isolated by others and named
as LATS2 (14), and we agree that it is likely that
kpm is another mammalian homologue of Drosophila
warts/lats. In the previous article, we showed
that the kpm protein has autophosphorylation activity in
vitro and undergoes M-phase-specific phosphorylation in
vivo (1) as has been reported with LATS1. It is unclear which
kinase is involved in the phosphorylation of kpm although the
Cdc2-cyclin B complex has been reported to phosphorylate LATS1 (15).
Overexpression of kpm resulted in an increase in cell population at the
S/G2/M phase. However, in contrast to LATS1, our knowledge
of kpm is still limited and its physiological function
remains largely unclear. Thus, it is to be determined whether kpm plays
a crucial role in regulation of cell growth as has been demonstrated
with LATS1. In the present study, we generated HeLa-derived stable
transfectants of kpm based on the tetracycline-responsive
expression system and analyzed the function of kpm in terms of cell
cycle progression and cell viability. Here we present data indicating
that kpm negatively regulates cell growth by inducing G2/M
arrest and apoptotic cell death through its kinase activity.
Cell Lines and Cell Culture Conditions--
HeLa Tet-Off cells
(16) were purchased from Clontech (Palo Alto, CA)
and maintained in Dulbecco's modified Eagle's medium (Invitrogen)
supplemented with 10% fetal calf serum (FCS) (Invitrogen) and 100 µg/ml G418 (Sigma Chemical Co.). For selection of double transfectants and induction of gene expression, tetracycline-free FCS
(Clontech) was used.
Generation of Tetracycline-responsive Gene-inducible Cell
Lines--
The hemagglutinin-A
(HA)1-tagged wild-type
kpm cDNA and its kinase-dead mutant described previously
(1) were recloned into pTRE vector (Clontech)
downstream of the tetracycline-responsive promoter to make pTRE-kpm-wt
and pTRE-kpm-kd, respectively. pTRE-Luc control response plasmid (16)
was obtained from Clontech. HeLa Tet-Off cells were
transfected with pTRE-kpm-wt, pTRE-kpm-kd, or pTRE-Luc together with
pTK-Hyg (Clontech) by electroporation using a Gene
Pulser (Bio-Rad Laboratories, Hercules, CA). After 2 days, transfected
cells were subjected to selection with 200 µg/ml hygromycin B
(Invitrogen) in the presence of 10 ng/ml doxycycline (Clontech). Hygromycin-resistant cell lines were
screened for induction of kpm-wt or kpm-kd expression upon removal of
doxycycline by Western blotting. Induction of luciferase activity in
pTRE-Luc-transfectants was confirmed with the luciferase assay kit
(Promega, Madison, WI) and luminometry (Berthold Australia Pty Ltd.,
Bundoora, Australia).
Cell Viability Analysis by MTT Assay and Trypan Blue Dye
Exclusion--
Both adherent and non-adherent cells were harvested by
trypsinization, and the viable cell number as well as the cell
viability was measured by microscopic examination with trypan blue dye
exclusion. Cellular proliferation was measured by reduction of MTT,
which corresponds to living cell number and metabolic activity (17). Cells were thoroughly washed, plated at 5×104 cells/well
in 24-well plates and incubated with or without 10 ng/ml doxycycline
for various periods of time (for 1-5 days). 50 µl of 1 mg/ml MTT
solution (WST-8, Nacalai Tesque, Kyoto, Japan) was added to each well.
After 1 h of incubation, the absorbance of each well was measured
at 492 and 630 nm using a microplate reader Benchmark (Bio-Rad
Laboratories) according to the manufacturer's protocol.
Western Blot Analysis--
Cells were harvested and lysed in
TG-VO4 solution (18) (1% Triton X-100, 10% glycerol, 0.198 trypsin
inhibitor units (TIU) of aprotinin per milliliter of Dulbecco's
phosphate-buffered saline lacking divalent cations with fresh 100 mM Na3VO4) containing 0.1%
phenylmethylsulfonyl fluoride, 1× Complete protease inhibitors (Roche
Applied Science). After centrifugation, the supernatants were
collected, and the protein concentration of each cell lysate was
measured. Adjusted amounts of cell lysates were separated on 7.5 or
12.5% SDS-polyacrylamide gels and transferred onto Immobilon-P polyvinylidene difluoride membranes (Millipore, Bedford, MA). After
blocking, the membranes were incubated for 1 h with the first
antibodies followed by incubation with peroxidase-conjugated second
antibody for 1 h. The protein bands were detected using the ECL
detection system (Amersham Biosciences) according to the manufacturer's instruction. The antibodies used for Western blotting were mouse anti-HA monoclonal antibody (12CA5) (Roche Applied Science),
rabbit anti-Cdc2 polyclonal antibody (Santa Cruz Biotechnology, Santa
Cruz, CA), rabbit anti-cyclin B polyclonal antibody (Santa Cruz
Biotechnology), a specific anti-phospho-Cdc2-Y15 rabbit polyclonal antibody (Cell Signaling, Beverly, MA), rabbit anti-Cdc25C
polyclonal antibody (Cell Signaling, Beverly, MA), and goat anti-actin
polyclonal antibody (Santa Cruz Biotechnology).
Cell Cycle Analysis--
kpm-wt-, kpm-kd-, and Luc-inducible
HeLa Tet-Off cell lines were cultured in medium without doxycycline for
the indicated periods of time, harvested and washed twice with ice-cold
phosphate-buffered saline (PBS) containing 0.1% glucose. Cells were
then fixed with 70% ethanol for 1 h and incubated in 1 ml of PBS
containing 50 µl/ml of propidium iodide (Sigma), and 66 units/ml
RNase (Invitrogen) on ice for 30 min. DNA content analysis was
performed by a FACScan with CellQuest software (BD Biosciences).
Cell populations at G2 and M phases were distinguished by
the reactivity with mitotic protein monoclonal 2 (MPM-2 mouse
monoclonal antibody) (Upstate, Waltham, MA) as described (19-21). In
brief, cells were fixed in 70% methanol and stained with MPM-2
antibody followed by incubation with (Fab')2 fraction of
FITC-conjugated goat anti-mouse IgG (BIOSOURCE,
Camarillo, CA). After washing, cells were incubated with propidium
iodide for DNA staining and then analyzed by two-color flow cytometry
using the FACScan (BD Biosciences). MPM-2 reactive cells were
considered to be at the mitotic phase, and the percentage of this
population represented the mitotic index.
In Vitro Kinase Assay of the Cdc2-Cyclin B Complex--
Kpm-wt-,
kpm-kd-, and Luc-inducible HeLa Tet-Off cell lines were cultured for
48 h with or without doxycycline, and then treated with 0.5 µg/ml nocodazole (Sigma) for the last 24 h (11, 22, 23). Both
adherent and non-adherent cells were harvested by trypsinization,
washed three times with PBS ( TUNEL Assay--
Apoptosis was measured by TUNEL (24). HeLa
Tet-Off-kpm-wt, -kpm-kd, or -Luc cells were cultured for 5 days without
doxycycline and then subjected to TUNEL assay using the FlowTACS FITC
kit (Trevigen, Gaithersburg, MD). TUNEL+ cells were
quantified by flow cytometric analysis.
DAPI Staining--
To confirm the findings of TUNEL assay,
apoptotic cells were also detected by DAPI staining (25). Cells were
washed with PBS ( Establishment of Stable Transfectants of kpm-wt, kpm-kd, and
Luciferase under the Control of the Tetracycline-responsive
Promoter--
HeLa Tet-Off cells were transfected with either
pTRE-kpm-wt, pTRE-kpm-kd, or pTRE-Luc together with pTK-Hyg and
subjected to selection with hygromycin B. Several stable transfectant
lines of the three genes were expanded and screened for efficient gene induction by the removal of doxycycline. Representative transfectants of each of the three genes were compared with parental cells for the
gene expression in the absence or presence of doxycycline. Western blot
analysis showed that the representative lines of HeLa Tet-Off-kpm-wt
and -kpm-kd were induced to express a large amount of kpm protein when
cultured without doxycycline (Fig. 1A). Expression of kpm was
detected after 12 h and reached maximal levels after 48 h,
dependent on the concentrations of doxycycline (Fig. 1B).
Based on scanning densitometry, removal of doxycycline resulted in more
than 100-fold induction of kpm-wt or kpm-kd by probing with anti-kpm
polyclonal antibody, which recognize both endogenous and exogenous kpm
(data not shown). Likewise, high levels of luciferase activity were
induced in a representative HeLa Tet-Off-Luc upon the removal of
doxycyline (data not shown).
Overexpression of kpm Inhibits Cell Growth--
In order to
explore the biological function of kpm, we first examined the effects
of overexpression of kpm on cell viability and proliferation. HeLa
Tet-Off-kpm-wt, -kpm-kd, and -Luc cells were switched into the culture
without doxycycline, and the viable cell number was counted daily by
trypan blue dye exclusion. As shown in Fig.
2A, induction of kpm-wt
expression resulted in a decline in viable cell number after 2 days
compared with non-induced culture. In contrast, cell growth of HeLa
Tet-Off-kpm-kd as well as -Luc was not affected by expression of these
genes. In accordance with this, the MTT assay also showed that
overexpression of kpm-wt suppressed cell proliferation while that of
kpm-kd or luciferase had no effect (Fig. 2B). These results
indicate that kpm is involved in either cell cycle progression or cell
viability, and negatively regulates cell growth. Since overexpression
of kpm-kd had no effect on cell viability or proliferation as that of
luciferase, it is suggested that anti-proliferative effect of kpm is
dependent on its kinase activity and kd mutant does not function as a
dominant negative form at least in this particular assay system.
Overexpression of kpm Induces G2/M
Arrest--
Since overexpression of kpm-wt resulted in inhibition of
cell proliferation and a decline in viable cell number, a cell cycle arrest was suspected to have occurred. To determine at which stage of
the cell cycle cells were arrested, we performed the cell cycle analysis in the three transfectant lines upon removal of doxycycline. As shown in Fig. 3A,
overexpression of kpm-wt induced an increase in cell population in
G2/M phase and a decrease in cell population in
G1 phase compared with non-induced culture. In contrast,
there was no difference in the profile of cell cycle progression
between non-induced and induced overexpression of kpm-kd or that of
luciferase. To further analyze the kpm-induced cell cycle arrest and
determine whether it was a G2/M transition arrest or a
mitotic arrest, we performed scoring of mitotic index by MPM-2 assay
that had long been used to identify mitotic cells. Overexpression of
kpm-wt increased the cell proportion in G2/M phase as has
been shown but with no significant changes in cell proportion in
mitotic phase expressing MPM-2 antigen (Fig. 3B), indicating
that overexpression of kpm-wt resulted in a G2/M transition
arrest.
Kpm Negatively Regulates the Kinase Activity of the Cdc2-Cyclin B
Complex--
Since the overexpression of kpm induced cell cycle arrest
at the G2/M boundary, we next examined whether
overexpression of kpm negatively regulated the kinase activity of the
Cdc2-cyclin B complex. In parallel with the gene induction, cells were
synchronized in prometaphase and metaphase by the nocodazole method.
Western blotting with whole cell lysates showed that induction of
kpm-wt, kpm-kd, or luciferase did not affect the total amounts of Cdc2 or cyclin B (Fig. 4A).
Likewise, there was no particular difference in the amount of Cdc2
co-immunoprecipitated with cyclin B between non-induced and induced
cells. However, the Cdc2-cyclin B complex of kpm-wt-induced cells
showed much lower histone H1 phosphorylation acitivity than that of
non-induced cells, while induction of kpm-kd or luciferase had no
effect on the kinase activity (Fig. 4B). We repeated these
experiments three times and obtained similar results. Synchronization
by the double thymidine block did not work well with HeLa Tet-Off cells
and gave only incomplete results (data not shown), which were
nevertheless consistent with what we observed by the nocodazole method.
As has been described elsewhere (26, 27), Cdc2 co-immunoprecipitated
with cyclin B consisted of a doublet of bands of which the upper one
represented the hyperphosphorylated inactive form, and the lower one
represented the dephosphorylated active form. As shown in Fig.
4B, the proportion of the hyperphosphorylated inactive form
of Cdc2 was increased in kpm-wt-induced cells, which was also
demonstrated by Western blotting using a specific anti-phospho-Cdc2-Y15 antibody. These data suggest that Cdc2 bound to cyclin B remained or
was rendered inactive by the phosphorylation at Thr-14 and Tyr-15,
which seems to be the major mechanism of the G2/M
arrest.
Overexpression of kpm Affects the Phosphorylation Status of
Cdc25C--
We next investigated the possible involvement of Cdc25C in
the down-regulation of the Cdc2-cyclin B kinase. It is known that in
SDS-PAGE Cdc25C consists of an 85-kDa band of the hyperphosphorylated form with the highest phosphatase activity and a doublet of 60- and
57-kDa bands, which represent the Ser-216-phosphorylated inactive form
and the dephosphorylated form with weak phosphatase activity, respectively (28, 29). In parallel with gene induction, cells were
synchronized in prometaphase and metaphase by the nocodazole method.
Western blotting with whole cell lysates showed that the 85-kDa
hyperphosphorylated active form as well as the doublet of Cdc25C were
present in luciferase- or kpm-kd-induced cells as described above. In
contrast, it was noted that the mitotic hyperphosphorylated form was
almost undetectable and conversely the Ser-216-phosphorylated inactive
form (the upper band of the doublet) was increased in kpm-wt-induced
cells (Fig. 4C). These data suggest that Cdc25C remains or
is rendered inactive by overexpression of kpm-wt resulting in the
decrease in the activity of dephosphorylating Cdc2, which seems to be
one of mechanisms of the inactivation of the Cdc2-cyclin B kinase.
Overexpression of kpm Induces Apoptosis after
G2/M Phase Arrest--
Because many tumor
suppressor genes are known to inhibit cell growth by inducing apoptosis
as well as cell cycle arrest, we examined whether this was also the
case with kpm. In fact, induction of apoptosis by kpm was already
suggested by cell cycle analysis in which an increase in
sub-G1 phase cells with a DNA content less than 2 N was
observed after an elongated induction of kpm-wt for more than 4 days
(Table I). To demonstrate that this
population was generated as a result of apoptosis, cells after kpm
induction were first subjected to TUNEL assay. As shown in Fig.
5A, an increase in
TUNEL-positive cells was clearly detectable in kpm-wt-induced but not
non-induced cells. Induction of kpm-kd or luciferase elicited no change
in TUNEL-positive cells. Presence of apoptotic cells was confirmed by
DAPI staining and immunofluorescence microscopy. Induction of kpm-wt
resulted in chromatin condensation and segregation characteristics of
apoptotic cells whereas that of kpm-kd or luciferase did not (Fig.
5B).
We previously reported the molecular cloning of kpm,
which encodes a putative human serine/threonine kinase homologous to warts/lats, a Drosophila tumor
suppressor (1). Prior to our article, Tao et al. (11)
described a human as well as mouse homologue of
warts/lats named LATS1 that could
functionally compensate the defect of warts/lats
in Drosophila. In contrast to LATS1 that has been
extensively studied, the function of kpm remains largely unknown. It is
to be determined whether kpm has similar or unique function compared
with LATS1. In the present study, we established HeLa-derived stable
transfectants of wild-type kpm (kpm-wt), a kinase-dead mutant of kpm (kpm-kd), and
luciferase under the control of tetracycline-responsive promoter in
order to define the biological function of kpm. Using this system (16,
30), we demonstrated that overexpression of kpm-wt resulted in
suppression of cell proliferation due to cell cycle arrest in
G2/M phase and subsequent apoptotic cell death.
Cell cycle analysis combined with MPM-2 assay clearly showed that
overexpression of kpm-wt induced a cell cycle arrest by blockade of
G2/M transition rather than delaying progress of mitosis. Consistent with this, we showed that the histone H1 kinase activity of
the Cdc2-cyclin B complex was markedly diminished in kpm-wt-induced cells. Furthermore, Cdc2 bound to cyclin B remained or was rendered phosphorylated at Tyr-15 in kpm-induced cells. It is well known that
the transition between the G2 phase and mitosis is
regulated through inhibitory phosphorylation of the Cdc2 kinase (31,
32). Since overexpression of kpm-wt did not change the protein levels of Cdc2 and cyclin B in the whole cell lysates as well as in the immunoprecipitates by anti-cyclin B, it seems likely that
overexpression of kpm led to a cell cycle arrest at G2/M by
increasing the ratio of the hyperphosphorylated inactive form of Cdc2.
We examined whether Cdc25C was involved in the inactivation of Cdc2
because it is established that phosphorylated Cdc2 is dephosphorylated
by this dual-specific phosphatase. Western blot analysis showed that
overexpression of kpm-wt resulted in a marked decrease in the
hyperphosphorylated active form of Cdc25C and an increase in the
Ser-216-phosphorylated inactive form. Considering that 14-3-3 proteins
bind to phosphoserine 216 of Cdc25C and translocate it from the nucleus
to the cytoplasm, the overall phosphatase activity of Cdc25C should be
strongly down-regulated in kpm-wt-induced cells, which seems to be one
of the mechanisms of the increase in phosphorylated inactive form of
Cdc2. Although we do not exclude other possible mechanisms for the
kpm-induced cell cycle arrest in G2/M phase, it is certain
that the kinase activity of kpm itself plays the central role in such a
putative phosphorylation-dephosphorylation cascade, because kpm-kd had
no effect.
LATS1 has also been reported to inhibit cell growth and induce cell
cycle arrest in G2/M (33, 34). However, the mechanism of
the cell cycle arrest in LATS1 overexpression is different from that of
kpm described here. According to Tao et al. (11) LATS1 could
associate with Cdc2 and competitively inhibit the binding of cyclin B
to Cdc2, which resulted in a decrease in kinase activity of the
Cdc2-cyclin B complex. In addition, ectopic expression of LATS1 in
MCF-7 cells has been reported to induce specific down-regulation of
protein levels of cyclin A and cyclin B, while no effect was found on
cyclin E, Cdc2, CDK2, p27KIP, and p21CIP levels
(34). The discrepancy between kpm and LATS1 may be simply because these
two molecules are distinct from each other. The experimental systems
were also different, in which we used a tetracycline-responsive gene
expression system in HeLa-derived cells (30, 35) whereas LATS1
overexpression was induced by transduction of fibroblasts and other
cancer cells using adenovirus vectors (33, 34). We do not exclude the
possibility that kpm has the capacity to associate with Cdc2 although
we have not been able to demonstrate the association of these molecules
in vivo. However, the data presented here clearly indicated
that, at least in this system and in HeLa cells, the cell cycle arrest
at G2/M transition is not mediated by the competitive
inhibition of binding between Cdc2 and cyclin B but rather by the
increase in the ratio of phosphorylated inactive form of Cdc2 bound to
cyclin B. Thus, the present study has revealed the presence of a novel
pathway of G2/M regulation through kpm and the Cdc2-cyclin
B complex.
It is likely that the function of kpm is not restricted to the
regulation of the Cdc2-cyclin B kinase. In fact, we showed that
overexpression of kpm induced apoptotic cell death after cell cycle
arrest. With regard to this, our preliminary experiments have suggested
that expression of Bcl-2 protein is specifically downregulated after 3 days of kpm-wt induction (data not shown) although the signaling
cascade leading to apoptosis from kpm overexpression remains unclear.
In particular, the relationship between the cell cycle arrest and the
induction of apoptosis needs to be investigated. On the other hand, a
new cytoplasmic protein named salvador (hWW45, a WW domain
containing-gene) has recently been described that interacts with LATS1
to cause both cell cycle arrest in G1/S as well as
G2/M and apoptotic cell death (36, 37). kpm also has a
PPXY motif (1, 36, 37) that is predicted to interact with WW
domain, and actually we have recently found that salvador could be
coimmunoprecipitated with
kpm.2 Further studies are
required to molecularly define the function of kpm in terms of cell
cycle regulation as well as induction of apoptosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) for thorough removal of nocodazole,
and cultured without nocodazole for 50 min. Fluorescent microscopy of
DAPI-stained cells indicated that most cells were in prometaphase and
metaphase at this time point. Cells were harvested by trypsinization,
washed three times with PBS (
), and lysed with TG-VO4
solution containing 0.1% phenylmethylsulfonyl fluoride, 1× Complete
protease inhibitors. The protein concentrations of the cell lysates
were measured and adjusted equally. A part of each cell lysate was used
for the study of whole cell expression of Cdc2, cyclin B, Cdc25C, and
-actin by Western blotting. The rest of the lysate was subjected to
immunoprecipitation by anti-cyclin B monoclonal antibody (Santa Cruz
Biotechnology) and protein G-Sepharose (Amersham Biosciences). Half of
the immunoprecipitate was used to monitor Cdc2 co-immunoprecipitated
with cyclin B by Western blotting. The Cdc2 fraction phosphorylated at
Tyr-15 was detected by a specific anti-phospho-Cdc2-Y15 antibody. The
remaining immunoprecipitate bound to beads was used for in
vitro immune complex kinase assay as follows (18). The
precipitated beads were washed five times with TG-VO4
solution and once with kinase buffer (20 mM Tris-HCl, pH
7.5, 10 mM MgCl2, 1 mM EGTA) and
incubated in 20 µl of reaction buffer (20 mM Tris-HCl, pH
7.5, 10 mM MgCl2, 1 mM EGTA, 2 mM dithiothreitol, 1 mM
Na3VO4, 0.1 mM ATP, 10 µCi of
[
-32P]ATP, 5 µg of histone H1) at room temperature
for 20 min. 20 µl of 2× Laemmli sample buffer was added and boiled
for 5 min to stop the reaction. The phosphorylation of histone H1
(Sigma) was examined by separating the samples on a 15% SDS-PAGE gel
and autoradiography.
), transferred to 1.5-ml microtubes, and fixed with
1% glutaraldehyde at room temperature for 30 min. After washing with
PBS (
), cells were resuspended in 20 µl of PBS (
) and mixed with
5 µl of 10 µl/ml DAPI (Sigma). Cell suspensions were mounted on
slide glasses and subjected to fluorescence microscopic examination.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Conditional expression of kpm protein in HeLa
Tet-Off-kpm-wt and -kpm-kd. A, HeLa Tet-Off-kpm-wt and
-kpm-kd cells were cultured without doxycycline for 48 h and
subjected to Western blotting using anti-HA mAb. B, Western
blot analysis of kpm expression in HeLa Tet-Off cells after culture
with serial dilutions of doxycycline for 48 h. The same membrane
was reprobed by anti- -actin Ab as an internal protein control.
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Fig. 2.
Effect of overexpression of kpm-wt on cell
growth. After removal of doxycycline, cell growth was monitored by
viable cell counting (A) and MTT assay (B).
Viable cell number was counted in duplicate by trypan blue dye
exclusion. MTT assay was performed in quadruplicate, and the mean
values ± S.D. at 492 and at 630 nm were measured. The relative
values of induced cells to those of non-induced cells were plotted in
the graph. Three independent experiments were done, and the data of a
representative experiment are shown.
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Fig. 3.
Cell cycle analysis by propidium iodide
staining and MPM-2 assay. A, HeLa Tet-Off-kpm-wt cells
were cultured with (left) or without doxycycline
(right) for 3 days and harvested for DNA content analysis.
Cells were stained with PI and subjected to flow cytometric analysis
using a FACScan. Three independent experiments gave similar results and
a histogram of a representative experiment is shown. B, to
distinguish whether kpm-wt blocks G2/M transition or delays
progress of mitosis, mitotic index was measured in asynchronized
kpm-wt-induced and non-induced HeLa Tet-Off cells. Flow cytometric
analysis clearly differentiates G1 phase cells with low PI
: DNA content = 2 N and low MPM-2 expression, S phase cells with
PI : 2 N < DNA content < 4 N and low MPM-2 expression,
G2 phase cells with high PI : DNA content = 4 N and
low MPM-2 expression, and mitotic phase cells with high PI : DNA
content = 4 N and high MPM-2 expression. This assay was repeated
three times and gave similar results. Apoptotic cells and fragmented
cell debris that appeared in the sub-G1 region were not
included in these assays.
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Fig. 4.
Effects of overexpression of kpm on the
Cdc2-cyclin B complex and its kinase activity. A,
Western blot analysis of Cdc2 and cyclin B in whole cell lysates of
non-induced or induced HeLa Tet-Off-Luc, -kpm-wt, and -kpm-kd 50 min
after removal of nocodazole. Cdc2 consists of a doublet of bands of
which the upper band presumably represents the
phosphorylated form, and the lower band represents the
dephosphorylated form. B, immunoprecipitation of the
Cdc2-cyclin B complex and histone H1 kinase assay. The
immunoprecipitates by anti-cyclin B were blotted by anti-cyclin B,
anti-Cdc2, or a specific anti-phospho-Cdc2-Tyr15 antibody. The
Cdc2-cyclin B complexes were assayed for histone H1 kinase activities.
The phosphorylation of histone H1 was visualized by separating the
samples on a 15% SDS-PAGE gel and autoradiography. These experiments
were repeated three times and gave similar results. C,
Western blot analysis of Cdc25C in whole cell lysates of non-induced or
induced HeLa Tet-Off-Luc, -kpm-wt, and -kpm-kd cells at 50 min after
removal of nocodazole. Cdc25C consists of 85-kDa hyperphosphorylated
band and a doublet of 60- and 57-kDa bands, which represent
Ser-216-phosphorylated inactive form and the dephosphorylated with weak
phosphatase activity, respectively. These experiments were repeated
three times and gave similar results.
Sub-G1 populations in Luc-, kpm-wt-, or kpm-kd-induced cells
View larger version (36K):
[in a new window]
Fig. 5.
Apoptotic cell death induced by
overexpression of kpm. A, HeLa Tet-Off-Luc (lower
left), -kpm-wt (lower middle), and -kpm-kd (lower
right) cells were cultured with or without doxycyline for 5 days
and then subjected to TUNEL assay. TUNEL+ cells were
quantified by flow cytometric analysis. The upper row
indicates positive controls of parental HeLa Tet-Off cells treated with
3 µM staurosporine (upper left) or DNA
nuclease (upper right). Dotted lines indicate the
histograms of non-treated or non-induced cells. MFI indicates the
increase in mean fluorescence intensity. The difference between kpm-wt
non-induced cells and kpm-wt-induced cells was significant using a
Student's t test (p < 0.0005).
B, to confirm the findings of TUNEL assay, cells were also
analyzed by DAPI staining and fluorescence microscopy. Parental HeLa
Tet-Off cells treated with staurosporine were included as a positive
control. Condensed and segmented nuclei were detected only in
staurosporine-treated cells and kpm-wt-induced cells. These experiments
were repeated three times and gave similar results.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. E. Nishida (Faculty of Science, Kyoto University) for helpful discussions and critical comments on the manuscript, Dr. T. Kondo for experimental suggestions in the in vitro kinase assay, and K. Fukunaga for technical assistance.
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FOOTNOTES |
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* This work was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology.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 correspondence should be addressed: Dept. of Hematology
and Oncology, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawaracho, Sakyoku, Kyoto 606-8507, Japan. Tel.:
81-75-751-4964; Fax: 81-75-751-4963; E-mail:
thori@kuhp.kyoto-u.ac.jp.
Published, JBC Papers in Press, March 5, 2003, DOI 10.1074/jbc.M211974200
2 Y. Kamikubo and T. Hori, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: HA, hemagglutinin; MTT, 3,-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; DAPI, 4,6-diamidino-2-phenylindole; TUNEL, Tdt-mediated dUTP nicked end-labeling; PI, propidium iodide; PBS, phosphate-buffered saline.
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REFERENCES |
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