Download Mitosis and Polyploid Cell Formation

ICANCER RESEARCH56, 3551-3559, August 1, 19961
A Brief Staurosporine Treatment of Mitotic Cells Triggers Premature Exit from
Mitosis and Polyploid Cell Formation'
Lisa L. Hall,2 John P. H. Th'ng,
Xiao Wen Guo, Raymond
L. Teplitz,
and E. Morton
Bradbury
Department ofBiological Chemistry, School ofMedicine, University of Ca4fornia, Davis. California 95616 (L L H., X. W. G., R. L T., E. M. B./; Bloomfield Centerfor Research
in Aging, Lady Davis Institute, Jewish General Hospital, Montreal, Quebec H3T 1E2, Canada (J. P. H. T.J; and Life Sciences Division, Los Alamos National Laboratories, Los
Alamos, New Mexico 87545 (E. M. B.]
ABSTRACT
At any point during the progression of many tumor types, cells can
develop a hyperploid DNA conteni Hyperploid tumors are signiflcantiy
more aggressive, with a higher growth rate and a poor patient prognosis.
Yeast genetics have implicated
three important
genes Involved in DNA
ploidy changes: cdc2, cycin b, and a specific inhibiter of the p34@2/cyc1in
B kinase, rumi. Mutations in these genes uncoupled the dependence of
mlttsis on DNA replication in the fission yeast, Saccharomyces pombe. It
was proposed that the inactivation of the mitotic kinase complex, p34@2/
cydlin
B, induces
a G1 state
wherein
the cells
re-replicate
their
DNA
without an intervening mitosis. We show in this report that treatment of
only M phase-arrested mouse cells, with the protein kinase inhibitor
stauros@ne,
@
induced polyploidy. Nocodazole-arrested
metaphase
FF210
cells were pulsed with 100 ng/ml of staurosporine for 1 h. This 1-h
treatment results in the inhibition of the mitotic p34@ kinase. The
inhibition ofthe mitotic kinases leads to a reduction in the histone Hi and
H3 mitotic-associated phosphorylations, chromosome decondensation,
and nuclear membrane reformation. When released into normal growth
medium, these cells are reset to a G1 state, re-replicate their DNA without
completing
InItOSiS, and become octaploid.
produce polyploidy but instead increases the susceptibility (predispo
sition) to ploidy changes following additional genetic or externally
induced changes in the cell cycle (13). Because polyploidy/hyper
ploidy can occur in the very early stages of cancer, the study of the
molecular control of the cellular mechanism(s) of polyploidy may be
pivotal to our understanding of the progression of cancer.
There are situations, however, during normal cellular growth and
differentiation when M phase is uncoupled from S phase. Successive
S phases must take place without an intervening or completed mitoses
in some polyploid mammalian tissue cell types (14—16).Conversely,
meiosis requires that two rounds of segregation take place without
intervening S phases to produce haploid gametes. These examples
imply that cells are naturally capable of uncoupling M and S phases
and contain appropriate control pathways for this process, possibly
through the controlled inactivation of the p34'@2
(17—22).
In Saccharomyces pombe, the cdc2, cyclin b, and rum 1 genes have
been shown to play a direct role in DNA ploidy change (17—20).The
cdc2 and cyclin b gene products constitute the eukaryotic growth
associated Hl or M phase kinase complex p34'@―@2/cyclin
B. The loss
of @34cdc2
kinase activity, either through complete degradation of the
p34cdc2
INTRODUCTION
It has been suggested that mutations that increase the frequency of
whole chromosome abnormalities (genetic instability) may be partic
ularly important to cancer progression (1, 3). The induction of
polyploidy in continually cycling cultured cells may induce genetic
instability in the daughter cells by facilitating the formation of viable
aneuploid cells (2) and may play the same role during tumor forma
tion. Over one-half of all solid tumors in humans demonstrate
a
hyperploid
DNA content (4, 5), which results in an increased aggres
siveness and poor patient prognosis (6—9).Studies of preneoplasias
have demonstrated that hyperploidy can even occur prior to cellular
in essentially
“normal―
cells (10—12). Because
ploidy
change can occur so early in oncogenesis, it suggests the involvement
of very few or possibly even one gene direcfly involved in ploidy
control. The loss of cell cycle checkpoint controls does not directly
Received 11/13/95; accepted 5t28/96.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18U.S.C.Section1734solelyto indicatethisfact.
I Supported
by Grant GM4589O
from the NIH USPHS,
Grants
F51O and ERWF51O
(to
E. M. B.) from the Department of Energy Program, and a grant from Lady Davis
Foundation, Jewish General Hospital (to J. P. H. T.).
2 To
whom
through
insufficient
expression
requests
for
reprints
should
be
addressed,
at Department
of
the
cyclin
B
3 1), sister chromatid separation (32—34), and cytokinesis (35—38).
Alterations in any or all of these events can lead to polyploidy,
further
suggesting that changes in cellular control of the p34'@―2kinase
activity, during or prior to mitosis, may control cellular mechanisms
involved in mammalian cell ploidy change (2). Recently, there have
been indications that the loss of the p34@2 kinase may be responsible
for polyploidy in some higher eukaryotes, including mammals
(21, 22).
Polyploidy can be induced in mammalian cells by the use of many
drugs, including inhibitors
(40—42), topoisomerases
of the spindle apparatus (39), deacetylases
(43), and kinases (44, 45). These drugs
induce a cell cycle arrest during 02 or M phase. After an extended
period of time (—24—30
h), the cells begin to enter G, without
undergoing a complete mitosis. The periodicity of cell cycle protein
expression is generally maintained, indicating that the loss of check
point controls on some unknown “clock
mechanism― allows normal
cyclic gene expression throughout the drug arrest (44). These drug
studies may examine loss of checkpoint controls (predisposition) and
not the gene(s) directly controlling mammalian cell ploidy change.
stsp,3 a microbial alkaloid, has been shown to be a potent inhibitor
of a wide range of kinases tested in vitro (46—50);however, this may
not be the case in vivo. Although stsp is considered a broad kinase
Biological
Chemistry, School of Medicine, University of California, Davis, CA 95616. Fax: (916)
752-3516; E-mail: [email protected].
of
of the inhibitor p2SmmI
link the loss of p34cdc2/cyclmnB kinase activity with the generation of
polyploidy in yeast.
The p34@@2kinase is highly conserved between yeast and higher
eukaryotes (23) and in mammalian cells may be required for a number
of mitotic functions additional to those in yeast. These include chro
mosome condensation (24—27),nuclear envelope disassembly (28—
(2).
transformation,
(18),
(17,20) in yeastmutants,leadsto polyploidy.Thesestudiesclearly
Two important events in the eukaryotic cell cycle are responsible
for the genetic fidelity of the daughter cells. The first, DNA replica
tion, correctly duplicates the genetic material and the second, mitosis,
ensures that each daughter cell receives a complete set of chromo
somes. These two phases are tightly coordinated such that the initia
tion of one is dependent on the completion of the other (1). In the
event that the checkpoint controls for these phases are compromised,
an abnormal or skipped mitosis could occur, resulting in polyploid cell
formation
protein
subunit (19), or through the overexpression
3 The
abbreviations
used
are:
stsp,
staurosporine;
MI,
mitotic
index;
RLF,
replication
licensing factor.
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STAUROSPORINE
INDUCESPREMATUREMETAPHASEEXIT
inhibitor in vitro, normal cells have only two stsp arrest points during
the cell cycle, in G1 and 02 (51—53),and most transformed cells have
only the G2 arrest point (45, 52). This suggests that there may be
fewer cell cycle kinases sensitive to stsp in vivo than was previously
thought, and that only the mitotic kinases, responsible for the G2-M
transition, remain sensitive to stsp in transformed cells.
Here we report that a 1-h pulse of stsp administered to mouse cells
arrested
at metaphase
leads
to polyploidy
when
released
from
the
rylation
was
determined
by autoradiography
after
separation
of a 17.5%
SDS-PAGEgel.
Flow
Cytometric
Analysis.
Cells
treated
as described
above
were
har
vested by centrifugation and washed once with PBS before fixation in 70%
ETOH.Cells were stainedwith mithramycinA (Pfizer,Inc.) and analyzedon
a FMF II single argon laser system. All flow analysis was carried out at the
National Flow Cytometry and Sorting Resources at the Los Alamos National
Laboratory Life Sciences Division.
drug. This premature exit from mitosis into G1 may result from the
inhibition
of the p34cdc2 kinase
during
mitosis.
Our results
suggest
that in mammalian cells, the reset of mitotic cells to G@phase may be
controlled by the relative activity of the p34'@2 kinase and the
resulting changes in substrate phosphorylations.
MATERIALS AND METHODS
RESULTS
The Induction ofPolyploidy by stsp Pulse Treatment. stsp treat
ment has been shown to inhibit the p34'@2 kinase in vivo.5 The effect
of stsp inhibition on the p34@2 kinase in metaphase-arrested mouse
cells and the resulting cell cycle progression was studied. The mouse
mammary
tumor cell line FF210
was synchronized
through
sequential
Cell Culture Conditions. The mouse mammarytumorcell line, FF210, cell cycle arrests, with a final arrest in M phase with nocodazole
(typically, a MI of between 65 and 75% was obtained for FF210
was maintained at a density of about S X l0@ cells/mi in RPMJ 1640
supplemented with 10% calf serum. The cell density was increased to 1 X l0@ cells). Half of the nocodazole-arrested cells were treated with a 1-h
cells/ml for all experiments.
pulse of 100 ng/ml stsp, while the other half remained in nocodazole
Cell Synchronization.The synchronization
protocolwas basedon that only. Both cell populations were then washed twice in PBS and
described in Th'ng et a!. (54). The asynchronously
growing cells were arrested
released into drug-free medium. Samples were taken every 2 h fol
in G1 with isoleucine-deficient
RPM! 1640 supplemented with 10% heat
lowing release and were analyzed by flow cytometry (Fig. 1).
inactivated and dialyzed calf serum for 16 h (55). The cells were released into
Microscopic examination indicated that the MI for the control,
normal growth medium containing aphidicolin (2.5 @g/ml)
for 9 h to arrest the
cells at the G1-S boundary. Then the cells were washed once with PBS and nocodazole-arrested cells in Fig. 1 was —70%prior to release. Flow
cytometric analysis showed that over 70% of the control cells had
transferred to normal growth medium for 18 h with nocodazole (50 ng/ml)
added for the last 12 h, where cells with a mitotic index of 60—70%were entered G1 by 2 h after release (Fig. 1, row A), and by 6 h, over 95%
of the cells had passed through mitosis and entered G1, as shown by
obtained. Since the cells take —12.9h to traverse the cell cycle from the G1-S
transition to metaphase, the majority of the cells are arrested at metaphase for
the appearance of cells with a 2C DNA content. Microscopic exam
only about 5—6h before treatment.4
ination of the cells shortly after the release from nocodazole
arrest
stsp
Treatment.
Synchronized
cells arrested
in M phase
with
100 ng/ml
nocodazole were treated with 100 ng/ml (0.2 ,xM)of stsp (Sigma Chemical
Co.) for 1 h while still in nocodazole.Subsequently,they were washedtwice
with PBS and then transferred to drug-free medium. At specific time intervals,
as indicated in the figures, samples were taken for analysis. Control cells that
were similarly arrested in M phase with nocodazole were mock treated and
released
into drug-free
media.
Samples
were then taken
in parallel
Index.
Cells
were
fixed
either
with
3.7%
formaldehyde
a reduction
in the percentage
of cells
with chromosomes
aligned at the metaphase plate (MI) and the appearance of cells in
anaphase and telophase. Flow analysis revealed that by 6 h after
release,
the control
cells had initiated
DNA replication,
as indicated
by a broadening of the G1 peak (Fig. 1, row A, filled arrow), and the
majority
with the
were in mid-S phase by 12 h, with no production
of polyploid
cells.
When 100 ng/ml of stsp were added to the nocodazole-arrested
stsp-treated cells.
Mitotic
revealed
in PBS
then stained with 0.1% Hoechst 33342 as described in Th'ng et a!. (54) or
FF210
stained with 0.1% Hoechst 33342 without fixation while cells were in normal
rapidly from 70% to less than 5% after a 1-h incubation. Microscopic
growth medium for 1 h. Mitotic indices were determined under a Leica
fluorescence microscope. A minimum of 200 cells were scored for each
sample. Only cells showing clearly defined chromosomes and absent nuclear
membranes were scored as mitotic cells (prometaphase or metaphase).
Histone Phosphorylation. Histones were extracted as described by Th'ng
et a!. (54). Cells were incubated in [32P)orthophosphoricacid 12 h prior to
harvesting. The cells were then collected by centrifugation, washed once with
ice-cold PBS, and lysed in nuclei buffer (0.25 M sucrose, 0.2 M NaC1, 10 mM
Tris (pH 8.0), 2 mM MgCI2,
1 mM CaCl2, and 1% Triton X-100].
Histones
were
cells, microscopic
examination
examination
of the cells shortly
tion in the percentage
showed
that the MI decreased
after stsp treatment
of cells with defined
revealed
chromosomes,
a reduc
an increase
in cells showing partially decondensed chromosomes, reforming nu
clei, and no cells in anaphase or telophase. These changes indicate that
stsp treatment causes a direct transition from metaphase morphology
to interphase morphology, similar to that described by Th'ng et al.
(54). Flow cytometric analysis of the cells released into a drug-free
medium (Fig. 1, row B) shows that 70% of the stsp-treated cells
then extracted with 0.4 N H2SO4and precipitated in 20% trichloroacetic acid.
The precipitate was washed with acetone, resolubilized in acid-urea gel sample
buffer (methylgreen; 8 M urea, 10% glycerol, and 5% acetic acid) and then
loaded onto a 12% acrylamide gel containing 8 Murea as described by Th'ng
et al. (54). Following electrophoresis, the proteins were stained with Coomas
remained tetraploid,
as shown by a 4C DNA peak, even after 4 h
incubation
in drug-free
medium.
The remaining
cells (30%) had
completed
mitosis and entered G1 phase (2C) 6 h after release,
sic Blue and dried. Autoradiography was performed with Kodak XAR film.
Kinase Assay. Cells were lysed in lysis buffer [0.1 M Tris (pH 8.0), 0.25 M
contrast,
NaC1,0.5% Triton X-100, I mMphenylmethylsulfonyl fluoride, 50 @tg/mi
each
of aprotinin and leupeptin, 1 mMNa3VO4 1 @M
okadaic acid, and 100units/mi
micrococcal nucleate] as described by Guo et a!. (56). Lysates were adjusted
to approximately I mg of protein to 500 @xl
lysis buffer for immunoprecipita
tion of the kinases. Lysates containing 1 mg of protein were used to immu
noprecipitate
p34cdc2 and then were assayed
for kinase
activity
using
initiated DNA replication by 8 h, and were in mid S phase by 12 h. In
over 50% of the cells in the tetraploid
population
started
to
re-replicate between 4 and 6 h following release (Fig. 1, row B, open
arrow), almost 3 h ahead of the 30% cells entering G@phase, and
these cells became fully octaploid (8C) by 12 h. In summary, these
flow cytometric analyses demonstrate that mitotic cells pulsed for 1 h
with 100 ng/ml stsp prematurely exit mitosis, and when released from
all drugs, re-replicate their DNA as polyploids. This experiment
[-y-32P]ATPand with histone Hl as substrate. The level of histone phospho
5 x.
‘w.Guo,
J. P. H. Th'ng,
R. Swank,
H. Y. Chen,
differentially inhibits the p34―2 and p33@
4 R. A.
Tobey,
unpublished
data.
and E. M.
Bradbury.
Staurosporine
kinases in mammalian cells, submitted for
publication.
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STAUROSPORINE INDUCES PREMATURE METAPHASE EXIT
A
Fig. 1. RowA, flowcytometricanalysisof no
Oh
2h
4h
6h
8h
lOh
codazole-arrested FF210 control cells that were
released into normal growth medium for 0-12 h
and produced no polyploid cells. arrow, initiation
@
of DNA replications. The first histogram (column
A, rowA) shows asynchronous FF210 cells. RowB,
flow analysis of FF210 cells that were synchro
A
,@[J
@.
EL
LL@
1,
r,
nir.ed at mitosis with nocodazole, treated with 100
ng/mlstspfor 1h, andthenreleasedintodnsg-free
@
medium forO—I2h showed generation of polyploid
cells. arrowhead, replicating cells. Row C, flow
analysis of FT2IO cells synchronized at mitosis by
an overnight treatment with nocodazole (18 h) and
B
L11 L,4 [4 ,
[email protected]@ L@
heldfor an additional12h in nocodazolefailedto
produce polyploid cells. arrow, no replication by
12h. RowD, flowanalysisof nocodazole-arrested
FI'2l0 cells treated with 100 ng/ml stsp for 1 h
before release back into nocodazole-contairnngme
@
dia still showed polyploidy by 12 h. arrow, repli
rated cell population after stsp-treated cells were
released back to nocodazole-containing medium.
C
I
Li
Li
L1 Li
LI@ L
The bars underthe histogramsindicateeither2C,
4C,or 8CDNAcontent.Thefirstbar corresponds
to the G, DNA contentof 2C, the secondbar
@
corresponds to the G2 DNA content of 4C, and the
lo.st bar corresponds to the second G2 (+) DNA
content of 8C.
D
[ UII @iiiL@IlL@[email protected]
L,.@,
(including all controls) was repeated five times, and the cell cycle
timing of all cell populations remained constant.
Microscopic Analysis of Polyploid Cells Induced by stsp Treat
ment. The chromosomes of the resultingoctaploid cells were exam
med for any obvious replication abnormalities
chromosomes
or fragments).
Nocodazole-arrested
(partially replicated
M-phase
treated with stsp for 1 h and then released into nocodazole-containing
medium for 24 h. This captures the octaploid cells in the following M
phase. Samples were taken during the initial metaphase arrest, after
the 1-h stsp treatment, and during the following metaphase arrest for
microscopic examination. The unfixed cells were stained with 0.1
cells were
mg/mi
Hoechst
33342
and examined
under
a fluorescence
micro
Fig. 2. @JfltiIl@11@were
treated with stsp to induce re-replication as described in Fig. lÀ.Nocodazole was added to arrest the cells at metaphase, and the chromosomes were
visualized under@i@k@i1I microscope after staining with Hoechst 33342. A and B show nocodazole-arrested mitotic untreated tetraploid Ff210 cells. A, cells stained with Hoechst;
B, the same cells
@[email protected] and D, nocodazole-arrested FF210 cells during a 1-h stsp treatment. C, DNA-stained cells; D, the phase contrast ofthese cells. E, the resulting
octaploid cells nii@tail in metaphase 24 h after release from stsp. F, some octaploid FF210 cells showing classic endo-reduplicated chromosomes. arrowheads, two paired re-replicated
sister chromosomes (end-reduplication).
3553
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STAL.JROSPORINEINDUCES PREMATURE METAPHASE EXIT
scope.
@
Fig. 2, A and B, shows
nocodazole-arrested
mitotic
(4C) cells
l7hr
prior to stsp treatment, displaying condensed chromosomes and no
nuclear membranes. This field shows an interphase nuclei as well
(30% of total population). A 1-h treatment with 100 ng/ml stsp results
in the decondensation
of the metaphase
chromosomes
LL
@1L
30 nglml LL
LL
_____
5OngImI@
Lk@_@i@
lOng/mi
Li
disorder of the chromosomes
upon decondensation.
This is consistent
with previous published data by Th'ng et al. (54). The stsp-induced
including
decondensation
other
mouse
also occurs in other cell lines tested,
cells
(FM3A
and
L-cells),
hamster
cells
(BHK), and human cells (HSF and HeLa). Octaploid (8C) FF210 cells
that were arrested in the following metaphase with nocodazole 24 h
after stsp treatment are shown in Fig. 2E. Although these octaploid
cells
@
were
generally
bigger
than
tetraploid
metaphase
cells,
(Fig. 2F, arrows).
stsp treatment
metaphase
chromosomes
when
100 ng!mI L J
mitosis
in the following
@i
arrested
in the following
II
M phase show multipolar
II
2c4c
Fig. 3. FF210
M
—@
2c4c
cells were synchronized
I
II
8c
I
2c4c
at S phase and then released
8c
into stsp concen
trations of 10, 30, 50, 70, and 100 ng/ml for up to 30 h. Samples were taken at 17, 24, and
30 h for analysis by flow cytometry. The G1 DNA content of 2C, the G2 DNA content of
phase and some endo-reduplication.
The octaploid peak is lost 27 h after release from stsp treatment in
the FF210 cells. Microscopic examination of the octaploid cells
traversing
LA.
of nocodazole-arrested
metaphase cells show immediate decondensation of metaphase chro
mosomes and production of interphase nuclei, which then re-replicate
their DNA-producing octaploid cells. These octaploid cells showed
typical
U
they
showed normal looking metaphase chromosomes (no fragmentation)
and no nuclear membranes. Further examination of the octaploid cells
showed endo-reduplication of the chromosomes in some of the octa
ploid cells
3Ohr
10 nglml
and the refor
mation of defined nuclear membranes, as shown in Fig. 2, C and D.
The reformation of interphase nuclei occurs during nocodazole arrest;
therefore, no separation of chromosomes (anaphase or telophase) was
observed under fluorescence microscope. However, we do observe
that some cells become multinucleated, which is most likely due to the
chromosome
24hr
4C,andthesecondG2(+) DNAcontentof 8Careindicatedon theXaxisof thelastrow
of histograms.
mitoses,
bipolar mitoses, and some cell death. This suggests that the loss of the
G2polyploid
peakmaybethrough
eithermultipolar
mitoses
orcell 17 h. When stsp concentrations reached 100 ng/ml, over 80% of the
cells were arrested in G2. Less than 5% of the total cell population
become polyploid by 30 h at these higher concentrations. In addition,
FF210 cells incubated in the presence of stsp for 30 h showed
extensive cell death, as evidenced by a gradual decrease in DNA
content (to the left of the 2C or 4C peaks) and by microscopic
death or both.
Nocodazole
Has No Effect
on Polyploidy
Production.
Nocoda
zole is known to induce polyploidy in some cell lines (39), either by
continuous
treatment (>20 h) or upon release from the drug due to
loss of checkpoint
controls like p53 (13). Therefore,
the potential
effects that nocodazole
may have on checkpoint
controls and the
examination.
in Fig. 1, row A, synchronized
FF210
cells, released
from overnight
nocodazole arrest into normal growth medium, do not form polyploid
cells. Continuous
incubation
of nocodazole-arrested
M phase cells in
nocodazole for an additional 12 h also failed to produce polyploid
cells
(Fig.
arrested
1, row C, double
arrow).
M phase cells were treated
In contrast,
when
population,
sufficient spindle
stsp Treatment
with stsp for 1 h and then released
checkpoints
Has Little
(13).
Effect
interesting
on Polyploidy
Production. Continuous incubation in 5—10ng/ml stsp has been
shown to induce polyploidy in a significant (40—50%)population of
MOLT-4 cells by 24 h (45). To determine the effect of long-term
treatment with stsp, FF210 cells were incubated in the presence of 10,
30, 50, 70, and 100 ng/ml stsp for up to 30 h. Cells incubated
in less
than 50 ng/ml stsp for 17 h do not seem to show a significant arrest
in G2-M, by flow cytometric
analysis
(Fig. 3). In MOLT-4
as low as 10 ng/ml,
some
resulting
in eventual
cell death.
Mitotic Requirement for Cells to Undergo Polyploidization. An
nocodazole
back into nocodazole-containing media, over 50% of the cells re
mained tetraploid and became octaploid by 12 h (Fig. 1, row D, line
arrow). These data suggest that nocodazole alone has no effect on the
polyploidy of these cell lines during this time period, and that these
cells maintain
Continuous
Even at stsp concentrations
cell death is seen. The absence of significant polyploidy suggests that
continuous stsp treatment in this cell line does not cause the majority
of the cells to skip M phase and re-replicate their DNA. Instead, it
may cause a complete cell cycle arrest in the majority of the cell
induction of polyploidy in the FF210 cell line was assessed. As shown
cells, only
these lower concentrations produced polyploid cells. However, the
FF210 cells treated with less than 50 nglml stsp may be slowly
traversing M phase without producing polyploid cells, even after 30 h
of continuous incubation. Only cells incubated in stsp concentrations
higher than 50 ng/ml display significant G2- or M-phase arrest by
observation
from
the data in Fig.
1, row B, is that the
number of cells arrested in metaphase (—70%MI) prior to the stsp
treatment corresponds to the number of cells that remain tetraploid
upon release. It is from these tetraploid cells that the octaploid cells
arise. The remaining
30% of the cells that traverse
a regular
mitosis
to
produce diploid cells corresponds to the percentage of cells that were
still in G2 at the time of stsp treatment. This suggests that the only
cells capable of becoming polyploid with stsp treatment are those in
M phase.
To verify whether only the mitotic cells are able to become
polyploid, FF210 cells were arrested in nocodazole for 9, 10, and 12 h
to produce cell populations with 30, 50, or 70% of the cells arrested
in M phase. These cells were then treated with 100 ng/ml stsp for 1 h
before release into drug-free medium for up to 10 h. Flow cytometric
analysis of treated cells with either 30, 50, or 70% MI show that by
10 h after release, the proportion of polyploid cells reflect the MI (Fig.
4, rows A, B, and C, respectively). The number of cells entering S
phase by 10 h with larger than 2C DNA content were roughly 20, 30,
and 45%, respectively. This experiment suggests there is a require
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@
B
STAUROSPORINE INDUCES PREMATURE METAPHASE EXIT
Ohr
@
4hr
A [j
6hr
8hr
. L
.L
lOhr
nocodazole-treated control cells (Fig. 5, row A) as shown in Fig. 1.
This indicates that these cells may initiate G1 directly upon release,
whereas the control cells must complete mitosis first. This is further
substantiated by using isoleucine-deficient (Ile) RPMI 1640, which
arrests FF210 cells in very early G1, possibly at the M-G1 transition
(55). When the stsp-treated, FF210 cells were released into 11e RPM!
1640, the tetraploid cells did not arrest, and re-replication continued to
take place at approximately the same time. This suggests that upon
release from stsp treatment, these cells re-enter the cell cycle after the
M-G1 transition, skip the Ile arrest point, and traverse a G1 phase
truncated by less than 1 h.
L
p34CdC2
Activity
Is
Lost
with
stsp
Treatment.
The
presence
of
stsp is known to dramatically
inhibit the activity of p34cdc2 and
minimally inhibit the p33'@―@
kinase in vivo.5 To follow the changes
C
I
ii
#@
2c4c
I
2c4c
___
___
in the p34cdc2 kinase activity after stsp treatment, the kinase corn
plexes were immunoprecipitated from cell lysates and assayed for
activity using histone Hi as substrate. Fig. 6 shows a graph of the
@@*i
*ff1@@M
2c4c 8c 2c4c 8c 2c4c 8c
Fig. 4. FF210 cells were arrested in M phase with nocodazole for 15, 17, and 19 h
before a 1-h treatment with 100 ng/ml sup (rows A, B, and C, respectively). The MIs for
each sample set before treatment were 32, 54, and 70%, respectively. Cells were released
into drug-free medium for up to 10 h and analyzed by flow cytometry. The G, DNA
content of 2C, the 02 DNA content of 4C, and the second G2 (+) DNA content of 8C are
indicated under the X axis of the last row of histograms.
12 h
A
p34cdc2
kinase
activity
compiled
from
densitometer
readings
of
four
different autoradiograms. These experiments show that the presence
of stsp results in a significant reduction in the activity of the p34@―@2
kinase to approximately 10% of the original (100%) M-phase level.
Interestingly, this kinase activity remains below 20% activity after
release from both drugs (Fig. 6, 0) for the duration of the experiment.
The activity of the p34cdc2 kinase in the nocodazole-arrested control
cells dropsto —5%of the original 100%level after releaseand
remains below 5% for the duration of the experiment (Fig. 6, •).
These data confirm that the ability of the immunoprecipitated p34cdc2
kinase complex to 32P-label histone Hl decreases dramatically with
stsp treatment
and remains
reduced
after release
from the drug for up
to 12 h. Metaphase cells treated for 1 h with 100 ng/ml of stsp results
@
in the reduction
@LjLj
2c4c
2c4c
2c4c
2c4c
2c4c
Fig. 5. FF210 cells were synchronized in G2 by incubation at the nonpermissive
temperature of 39°Cfor 18 h. The MI for these cells was 0%. These G2-arrested cells were
treated with 100 ng/ml sap for 1 h before release into normal growth medium at 32CC for
of p34cdc2 kinase
activity
to the approximate
levels
found in control cells following nocodazole release.
Histone Hi Hyperphosphorylation
Is Reduced following stsp
Treatment. Hyperphosphorylation of histone Hi and phosphoryla
tion of histone H3 have been correlated with the final stages of
0, 4, 8,and 12h.A,flowcytometricanalysisofan asynchronous
cellpopulation.Samples
were taken every 4 h for flow cytometric analysis. The same results were obtained for cells
arrested in G2 with Hoechst 33342 followed by step treatment. The G1 DNA content of
2C andthe G2DNAcontentof 4C are indicatedundertheX axisof all histograms.The
arrowhead indicates that there are no octaploid cells by 12 h after release.
ment for the cells to be in M phase for stsp to induce polyploidy, and
that G2 phase cells may not become polyploid with stsp treatment.
To test this possibility, FF210 cells were synchronized in G2 with
either 175 ng/ml of Hoechst 33342 (data not shown) or by incubation
at 39°Cbefore treatment with 100 ng/ml stsp (Fig. 5). The MI for both
types of G2 arrests
was 0%, confirming
the G2 arrest.
Following
.@
the
1 h incubation in stsp, these G2-arrested cells were released into
normal growth medium for up to 12 h, and samples were taken at
regular intervals. Flow cytometric analysis (Fig. 5) show that when
the cells were released from the 39°Cblock after stsp treatment, over
50% of the cells had divided and entered G1 phase by 8 h, with no
polyploidy
production.
Arrest
in G2 by either
Time (hr)
39°C or by Hoechst
Fig. 6. Nocodazole-arrested FI'210 cells were treated with 100 ng/ml stsp for I h
before release into normal growth medium for 12 h. Nocodazole-arrested FF210 cells, not
treated with stsp, were used as controls. The p34@2 kinase complex was immunopre
cipitated from the cell lysates using antibodies against a COOH-terminal peptide of the
human p34@2 protein. The activity of the kinase was assayed using 32Piand histone H1
33342 produced identical results. The data demonstrate that G2-phase
FF210 cells cannot bypass M phase and become polyploid following
stsp treatment.
Tetraploid Cells Traverse an Almost Complete G1 Phase before
Commencing Re-Replication. Normal G1 in the FF210 cell cycle
takes approximately 6.8 h to complete, whereas M phase takes close
to 30 mm.6 The initiation of DNA re-replication in the tetraploid (4C)
cells (Fig. 5, row B) occurs approximately 1 h in advance of the
as the substrate. The histone HI was then run on SDS-PAGE before autoradiography.
Densitometer readings on the autoradiograms of four repeated experiments were obtained.
The average and SDs for each time point are plotted for the nocodazole-released control
cells (•)and for the stsp-treated/released
cells (0). The first time point shows the initial
M phase percentage of kinase activity (—1 h time point). step was added for 1 h, or the
control cells were not treated for I h before a sample was taken (0 h time point). •,the
percentage of p34cdc2 kinase activity during a normal nocodazole release from 0—12h
alter release. 0, the percentage of p34@2 kinase activity in the cells released after a 1-h
6 R. A. Tobey
and E. M. Bradbury,
unpublished
data.
stsp treatment
(0—12 h).
3555
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STAUROSPORINE
INDUCESPREMATUREMETAPHASEEXIT
C/)
&&@1234
DISCUSSION
There have been numerous reports linking polyploidy with carci
nogenesis (4, 5, 60—63). A key initiating event in polyploid cell
production
Hil
@
I1I@@:
A
@II!9@
@,.,s
•wi
-@
I
H3@@i@II!.L@t
Fig. 7. FF210 cells were grown in the presence of 32Piand then arrested in G@with
11e RPMI 1640 (Lane Gi), in Ge-S with 2.5 mg/mI aphidicolin (Lane GuS), and in M
phase with 100 ng/mI nocodazole (Lane M). The metaphase-arrested cells were then
treated for 1 h with 100 ng/ml stsp (Lane stsp) and released into normal growth media for
1, 2, 3, and 4 h (Lanes 1—4,respectively). Histones were extracted and run on acid urea
gel electrophoresis before Coomassie staining and autoradiography. A, the Coomassie
stained gel; B, the resulting autoradiogram. arrowhead, hyperphosphorylated histone Hl
in mitotic cells.
chromosome condensation seen at mitosis (57—59).Since histone Hi
of p34'@@2, we studied
the changes
in histone
phosphorylations during and after release from stsp treatment.
During metaphase, the chromosomes are maximally condensed, the
nuclear membrane is clearly absent, histone Hi is hyperphosphory
lated (Fig. 7B, arrow), and histone H3 is phosphorylated. Previous
studies from this laboratory have shown that stsp (50—iOOng/ml)
added to nocodazole-arrested metaphase cells leads to decondensation
of chromosomes and dephosphorylation of histone Hi and H3 (54).
Fig. 7 shows a similar effect following a i-h treatment of metaphase
arrested cells with 100 ng/ml stsp. The phosphorylation levels on
histones Hi and H3 were reduced to interphase levels. These de
creases in histone Hi and H3 phosphorylations correlate directly with
the decondensation of the metaphase chromosomes, as illustrated by
a drop in MI from 70% to less than 5% by microscopic examination.
These cells showed diffuse chromatin and defined nuclear membranes
(Fig. 2, C and D). When the cells were transferred to regular growth
medium, there was a partial recovery of the hyperphosphorylated
forms of Hi and phosphorylation of H3 by 3 h (Fig. 7B). Since a small
increase in MI was also observed when these cells were released, the
increase in histone Hi and H3 phosphorylation was attributed to the
30% of G2 cells that enter mitosis after release. This is consistent with
the flow cytometric data in Fig. 1. A 1-h treatment with 100 ng/mi stsp
induces dephosphorylation of the histones Hi and H3 to the levels
seen during interphase concurrent with the decondensation of the
metaphase
chromosomes
to interphase
chromatin.
which
allows
the
mainly through the dual role the kinase plays in controlling the onset
of S and M phases (18, 19). Although p34cdc2 is not required for G1
or S phase progression in higher eukaryotes, a correlation was found
between the loss of p34@2 kinase activity, in some cycling plant and
mammalian cells, and the initiation of polyploid cell formation (21,
phosphorylations
@ H31
substrate
mitosis,
we show
that a brief
exposure
of metaphase
arrested mouse cells to stsp results in: (a) a complete and irreversible
loss of the p34cdc2 kinase activity (Fig. 6); (b) loss of the mitotic
B
is the putative
or abnormal
has
been
shown
tobe
involved
inpolyploid
cell
formation
(18—20
22). In this report,
@iII
Hil
must be an absent
cells to enter G1 with an increased DNA content (2). To date, the
gene(s) directly responsible for producing polyploidy in mammalian
cells remain to be identified. In yeast, the loss of the p34C@@@@2
kinase,
either through mutation or through the activity of its inhibitor p25tmm1,
This dephosphoryl
of histones
Hi and H3 (Fig. 7); (c) decondensation
of the metaphase chromosomes to interphase chromatin (Fig. 2); and
(‘0
reformation
ofnuclear
membranes
and
exit
from
metaphase
with
out completion of anaphase, telophase, or cytokinesis (Fig. 2). Upon
removal of nocodazole and stsp from the cells, they traverse an almost
complete G1 phase as polyploids (4C) before re-replicating their DNA
to become octaploid in the following G2 (Fig. iB).
Stsp has been shown to reversibly inhibit a wide range of kinases in
vitro (46—50), as well as a few kinases during interphase in vivo,
including p34cdc2 (52—54). However, in this report, we show that an
irreversible
inhibition of p34@@C2is produced in mitotic FF210 cells
(Fig. 6), which may involve cyclin B degradation. Commercially
available
cyclin
B antibodies
(from
Santa
Cruz Biotechnology,
Up
state Biotechnology, Inc., and PharMingen) failed to identify cycin B
protein in FF210 ceils. However, the same experiments performed in
mitotic HeLa cells resulted in an rapid degradation of cyclin B upon
stsp treatment.5 Since both cell lines produce irreversible p34cdc2
kinase inhibition upon stsp treatment, which result in polyploid cell
formation, we assume that stsp also induces cyclin B degradation in
the FF210 cell line. Additionally, in the FF210 cell line, there is no
change in the migration of the p34@2 protein on an SDS-PAGE gel,
indicating that the irreversible loss of the p34(x@c2
kinase activity is not
due to inhibitory phosphorylations on the protein.5 The continued loss
of the kinase after release from the drug suggests that further down
stream events may have been activated that triggered the “end―
of M
phase and entry to G1 , as found for a normal completed mitosis. It is
not clear whether the degradation of cyclin B after stsp treatment is
through the ubiquitin degradation pathway (64—67). This ubiquitin
mediated inactivation of p34@2/cyclin B usually triggers the exit of
M phase and the entry into G1 in a normal cell cycle. While the kinase
remains active, there is no entry into G1 (64, 65). If the loss of the
kinase activity upon completion of mitosis is the final downstream
checkpoint that triggers the entry into G1 phase during a normal cell
cycle, then the premature loss of the kinase activity during metaphase
by stsp treatment may be sufficient to trigger exit into G1, as shown
in this study.
Once the M-phase kinase, p34c@@2/cyclinB, activity is lost and the
cells are released from stsp, there are three possible outcomes for the
cells: (a) the re-formation of the metaphase chromosomes and the
completion of mitosis; (b) continuation of the cell cycle as G1
polyploid cells; or (c) or irreversible cell cycle arrest and death. Since
stsp-treated cells are now biochemically G1 cells, as regards kinase
activity (low p34cdc2 and low p34Cdk2 activity; Ref. 68) and have
ation of histones Hi and H3 persists after release from the drugs as the
cells continue to maintain interphase chromatin structure, by micro
interphase
chromatin
structure, an organized
re-condensation
of the
chromosomes may not be biochemically feasible. Furthermore, the
scopic examination.
3556
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STAUROSPORINEINDUCES PREMATURE METAPHASE EXIT
re-condensation
of disorganized
chromosomes
during the transition from M to G1 and may be reflected in the
may result in abnormal
condensation and apoptosis. Mammalian cells are capable of under
going multipolar mitoses during the following M phase and reconsti
tuting the diploid DNA content in the resulting daughter cells (2).
Thus, polyploidy may not even be permanent if all conditions remain
normal for the next mitosis, which is the case for the stsp-induced
polyploidy in the FT21O cell line. After the p34―2 kinase activity is
lost in the metaphase-arrested cells and they exit to interphase, the
most reasonable outcome for the cell is polyploid cell production, with
the possibility of re-establishing a diploid DNA content in the next
mitosis.
In this study, polyploidy could only be producedfrom metaphase
arrested FF210 cells but not from G2 phase-arrested cells treated with
stsp. The reason for this observation is not known. However, mam
malian
cells have been
shown
to require
the presence
of a RLF in
order to replicate (69—72).This requirement for the access of chro
matin to RLF was met in the M phase-arrested FF210 cells before stsp
treatment, enabling the cells to re-replicate after release. However,
once G2 or M phase cells (4C) have skipped or prematurely exited
mitosis, they are polyploid G1 phase cells (4C). Since these tetraploid
cells have been reset to G1, they are already polyploid prior to DNA
synthesis, which is where the RLF is required and where they re
replicate as polyploids. Therefore, RLF is not necessary for the G1
reset or required for the formation of polyploid cells. However, it is
needed
for those cells to re-replicate
02 phase cells differ
@
p33cdk2
p34cdc2
from
activities.
As
activity
in G1,
kinase
as polyploids.
mitotic
shown
5,
cells in their
by
G2,
Gu
and
et aL
M
relative
(68)
phase
p34c@k2 and
in HeLa
cells,
cells
approxi
are
the
mately 10, 5, 20, and 100%, respectively. On the other hand, p33@2
kinase activities in G1, 5, G2, and M phase cells are 10, 40, iOO, and
15%, respectively.
The stsp-induced
loss of p34CdC2 kinase activity
during mitosis was irreversible in the FF210 cell line. Previously, we
have shown that the effect of stsp treatment on G2 phase cells to be
completely reversible at the cellular level (54), and the inhibitory
effect of stsp on the p33Cdk2 kinase to be minimal in FF210 cells.5
Therefore 02 phase-arrested
recover from the treatment.
FF210 cells treated with stsp are able to
These G2 cells have high p33'@'@2activity
(68), which is less sensitive to stsp treatment,5 and would either
maintain or reconstitute the proper G2 phase p33@―@2
kinase activity
after release
@
from the drug, thereby
maintaining
the G2 kinase balance
(high p33@ and low p34CdC2;Ref. 68). These post-treated cells could
then activate the M-phase kinase at the proper time and proceed
normally through the G2-M transition without a G@reset and without
polyploid cell production. Conversely, the dramatic and irreversible
inhibition of the p34cdc2 kinase during mitosis, while the cells already
contain low p33Cdk2kinase activity (68), would produce a G1-phase
kinase balance (low p34@2 and low p33@@; Ref. 68). These cells are
then reset to G1, where they re-replicate their DNA as polyploid cells.
Thus, only mitotic cells contain the appropriate kinase balance such
that the inhibition of p34c@k2kinase would produce G1 phase kinase
activity, resulting in 01 phase-specific changes in the substrates and a
G1 reset.
The changing balance of kinase activities during the cell cycle may
phosphorylation
states
of the phase-specific
changes in the phosphorylation
as histones
and lamins,
substrates.
Thus,
the
states of the p34@2 substrates, such
may trigger
both the exit from metaphase
and
the reset to G1.
Many cell cycle-inhibitory drugs have been shown to induce
polyploidy in a number of mammalian cell lines after prolonged
exposure to the drug (39—45). These long-term cell cycle arrest
studies mainly follow the effects of cell cycle arrest on loss of
checkpoint controls between species or during cellular transformation
and not on gene(s) directly regulating ploidy change (74, 75). Cell
cycle checkpoint loss does not usually produce polyploidy directly but
instead predisposes the cell to ploidy changes following additional
genetic or externally induced changes in the cell cycle (e.g., the p53
checkpoint and spindle inhibition by drugs; Ref. 13). The production
of polyploidy through changes in the activity or abundance of gene
product(s) that are directly involved in the celluiar control of ploidy
change should not be dependent upon additional loss of checkpoint
controls. In the FF210 cell line, we tested for possible loss of common
cell cycle “clock―
checkpoints by prolonged incubation in M phase
with nocodazole (Fig. 1C) and G2 phase with stsp (Fig. 3). No
significant polyploidy was produced with these long-term treatments,
suggesting that the majority of the cell population may still maintain
sufficient cell cycle “clock―
checkpoints during G2 and mitosis. Ad
ditionally, although the FF210 cells had been exposed to nocodazole
for 12 h, they had been presynchronized, which means that the
majority of the population was arrested at metaphase for only 5—6h
before the brief 1-h treatment with stsp and the resulting polyploidy
production. Thus, there was no long-term (24—30h) cell cycle arrest
at metaphase, which makes it unlikely that the loss of a cell cycle
“clock―
checkpoint is involved. Instead, the induction of polyploidy
may be due to the inactivation of an important protein product directly
involved in the control of mammalian cell ploidy change, such as the
p34cdc2
kinase.
In conclusion, although stsp may be acting on an unknown
kinase that directly regulates mitotic and G1 phase control, our data
suggest that the premature inhibition of the p34@@2kinase alone
may be sufficient to trigger the G1 reset in mitotic cells. This
trigger for the G1 reset may be due to the premature loss of the
mitotic p34cdc2 kinase activity in a cell already containing G1
levels of the p33cdk2 kinase (68). Thus, the change from an M
phase to a G1-phase balance of kinase activities is reflected in the
phosphorylation
levels
of its phase-specific
substrates,
such
as
histone Hi and H3, nuclear membrane re-formation, and the state
of chromatin condensation. This change in substrate phosphoryla
tion may trigger entry into 0 . No other known kinase, active
during mitosis, controls as many M phase-specific activities known
to be involved in ploidy change mechanisms as p34cdc2. Addition
ally, this kinase has already been shown to be involved in ploidy
change in yeast and has been correlated with ploidy change in
maize endosperm and rodent parotid glands. A recent publication
has shown a direct correlation between loss of the p34―@2/cyclinB
kinase activity and megakaryocyte (4C) cell formation (76).
link the phases of the cell cycle with chromosomes through the
phosphorylation
states of histone
Hi and other substrates.
Since there
is a correlation between phosphorylation levels of some histones and
the condensation state of chromatin during the cell cycle (57—59),the
structural state of chromatin may also function as a cell cycle check
point
to prevent
the re-replication
of the DNA
or as a cell cycle
“clock―
controlling the timing of some phase-specific gene expression
(73). In this study, when the M-phase cells were treated with stsp, the
activity of the p34cdc2 kinase was reduced to approximately 10% of
the original level. This is similar to the drop in activity seen in cells
ACKNOWLEDGMENTS
We thank Carolyn Bell-Prince and Harry Crissman of the Los Alamos
National Flow Cytometry and Sorting Resources for the cell cycle analysis and
advice.
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A Brief Staurosporine Treatment of Mitotic Cells Triggers
Premature Exit from Mitosis and Polyploid Cell Formation
Lisa L. Hall, John P. H. Th'ng, Xiao Wen Guo, et al.
Cancer Res 1996;56:3551-3559.
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