Published OnlineFirst March 23, 2011; DOI: 10.1158/1535-7163.MCT-10-0809
Molecular
Cancer
Therapeutics
Therapeutic Discovery
Determinants of Mitotic Catastrophe on Abrogation
of the G2 DNA Damage Checkpoint by UCN-01
Kin Fan On, Yue Chen, Hoi Tang Ma, Jeremy P.H. Chow, and Randy Y.C. Poon
Abstract
Genotoxic stress such as ionizing radiation halts entry into mitosis by activation of the G2 DNA damage
checkpoint. The CHK1 inhibitor 7-hydroxystaurosporine (UCN-01) can bypass the checkpoint and induce
unscheduled mitosis in irradiated cells. Precisely, how cells behave following checkpoint abrogation remains
to be defined. In this study, we tracked the fates of individual cells after checkpoint abrogation, focusing in
particular on whether they undergo mitotic catastrophe. Surprisingly, while a subset of UCN-01–treated cells
were immediately eliminated during the first mitosis after checkpoint abrogation, about half remained viable
and progressed into G1. Both the delay of mitotic entry and the level of mitotic catastrophe were dependent on
the dose of radiation. Although the level of mitotic catastrophe was specific for different cell lines, it could be
promoted by extending the mitosis. In supporting this idea, weakening of the spindle-assembly checkpoint,
by either depleting MAD2 or overexpressing the MAD2-binding protein p31comet, suppressed mitotic
catastrophe. Conversely, delaying of mitotic exit by depleting either p31comet or CDC20 tipped the balance
toward mitotic catastrophe. These results underscore the interplay between the level of DNA damage and the
effectiveness of the spindle-assembly checkpoint in determining whether checkpoint-abrogated cells are
eliminated during mitosis. Mol Cancer Ther; 10(5); 784–94. 2011 AACR.
Introduction
Cyclin-dependent kinase 1 (CDK1) is one of the major
protein kinases for promoting mitosis. CDK1 is activated by binding to cyclin B1 and T-loop phosphorylation. Although cyclin B1 starts to accumulate from
S phase, the cyclin B1–CDK1 complexes are kept inactive by MYT1/WEE1-dependent phosphorylation of
CDK1Thr14/Tyr15. At the end of G2 phase, the stockpile of
inactive cyclin B1-CDK1 is activated by the CDC25
phosphatases (1).
At the end of mitosis, cyclin B1 is targeted to ubiquitinmediated degradation by APC/CCDC20 (2). Activation of
APC/CCDC20 is initiated only when all the chromosomes
have achieved bipolar attachment to the mitotic spindles.
This is ensured by a surveillance mechanism termed the
spindle-assembly checkpoint, which senses unattached
Authors' Affiliation: Division of Life Science, Hong Kong University of
Science and Technology, Clear Water Bay, Hong Kong
Note: Supplementary material for this article is available at Molecular
Cancer Therapeutics Online (http://mct.aacrjournals.org/).
K.F. On and Y. Chen contributed equally to this work.
Current address for K.F. On: Clare Hall Laboratories, Cancer Research UK,
Potters Bar, UK.
Corresponding Author: Randy Y.C. Poon, Division of Life Science, Hong
Kong University of Science and Technology, Clear Water Bay, Hong Kong.
Phone: 852-23588703; Fax: 852-23581552. E-mail: [email protected]
doi: 10.1158/1535-7163.MCT-10-0809
2011 American Association for Cancer Research.
784
kinetochores or the absence of tension between the paired
kinetochores (3). The underlying mechanism involves the
binding of the checkpoint machinery to unattached kinetochores, formation of diffusible mitotic checkpoint complexes, followed by the inhibition of APC/CCDC20 by
MAD2.
After DNA damage, entry into mitosis is inhibited by
the G2 DNA damage checkpoint. In essence, damaged
DNA activates sensors that facilitate the activation of
ataxia telangiectasia mutated (ATM) and ATM and
Rad3-related (ATR). ATM/ATR then activates CHK1
and CHK2, which in turn inactivate CDC25 and activate
WEE1, thereby increasing the inhibitory phosphorylation
of CDK1 (4).
Uncoupling of the G2 DNA damage checkpoint can
promote unscheduled activation of cyclin B1–CDK1 and
trigger a process termed mitotic catastrophe (5–8). Historically, the term was first used (9) to describe the
lethal phenotype associated with fission yeasts containing mutations that overactivate the mitotic engine (10).
Mitotic catastrophe is now generally defined as a mode
of cell death associated with inappropriate entry into
mitosis (11). Many cancer cells have a partially impaired
DNA damage checkpoint, thereby unable to maintain a
G2 arrest and eventually undergo aberrant mitosis. Failure of these cells to undergo mitotic catastrophe is
believed to contribute to genome instability and tumorigenesis.
Agents that inhibit the ATM/ATR–CHK1/CHK2 axis
can stimulate mitotic catastrophe and are exploited as
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Determinants of Mitotic Catastrophe
potential chemotherapeutic agents (12). For example, an
CHK1 inhibitor called UCN-01 (7-hydroxystaurosporine;
C28H26N4O4) can ablate the DNA damage checkpoint,
resulting in premature mitotic entry and cell death (13–
15). Several clinical trials that combine various DNA
damaging drugs with UCN-01 are currently under way
(16, 17).
Although remarkable progress has been made in our
understanding of the DNA damage checkpoint, the
mechanisms involved in checkpoint abrogation and mitotic catastrophe remained largely unresolved. In this
study, we aimed to elucidate the factors that influence
mitotic catastrophe during UCN-01–induced mitosis.
Using single cell analysis and time-lapse microscopy,
we found that abrogation of the ionizing radiation (IR)induced DNA damage checkpoint could trigger either
mitotic catastrophe or successful progression into G1. Our
data indicated that the dose of IR, the effectiveness of the
spindle-assembly checkpoint, and the particular cell line
are major determinants of mitotic catastrophe.
with retroviruses expressing histone H2B-GFP (a gift
from George Tsao, University of Hong Kong) in the
presence of 5 mg/mL of polybrene for 2 days. After 1
week, GFP-positive cells were sorted into 96-well plates
using a FACSAriaII flow cytometer (Becton Dickinson).
Unless stated otherwise, cells were treated with the
following reagents at the indicated final concentration:
blasticidin (5 mg/mL), caffeine (2 mmol/L), dihydrocytochalasin B (DCB; 10 mmol/L), doxycycline (1 mg/mL),
nocodazole (0.1 mg/mL), puromycin (1 mg/mL), RO3306
(Alexis; 10 mmol/L), UCN-01 (100 nmol/L), and ZM447439 (Selleck Chemicals; 2 mmol/L). Cells were
transfected with plasmids or siRNA using a calcium
phosphate method (23) or LipofectamineRNAiMAX
(Invitrogen), respectively. Cell-free extracts were prepared as described (24).
IR
IR was delivered with a caesium137 source from a MDS
Nordion Gammacell 1000 Elite Irradiator. Unless stated
otherwise, cells were irradiated with 15 Gy of IR.
Materials and Methods
Materials
All reagents were obtained from Sigma-Aldrich unless
stated otherwise.
DNA constructs and siRNA
MAD2L1BP (p31comet) cDNA was amplified by PCR
with 50 -GCCATGGCGGCGCCGGAGGCGG-30 and 50 CGGATCCTCACTCGCGGAAGCCTTT-30 ; the PCR product was cut with NcoI–BamHI and ligated into pUHD-P1
(18) to obtain FLAG-p31comet in pUHD-P1. The NcoI
fragment from FLAG-3C–cyclin B1(CD62)–EGFP (19)
was ligated into mRFP1 in pUHD-P3T (19) to create
mRFP1–cyclin B1(CD62) in pUHD-P3T. A puromycinresistant gene cassette was then inserted into the BamHI
site of the above constructs. Constructs expressing MAD2
shRNA and HA-MAD2 were generated as described (20).
Stealth siRNAs were obtained from Invitrogen.
Cell culture
HeLa cells were obtained from American Type Culture
Collection. No authentication was done by the authors.
The HeLa cell line used in this study was a clone that
expressed the Tet repressor (21). To generate anaphasepromoting complex/cyclosome (APC/C) reporter cells,
HeLa cells expressing histone H2B-GFP (green fluorescent protein; ref. 22) were transfected with mRFP1–cyclin
B1(CD62) in pUHD-P3T. To generate p31comet-inducible
expressing cells, HeLa cells were transfected with FLAGp31comet in pUHD-P1. The cells were then selected with
puromycin for about 2 weeks. Individual colonies were
isolated and propagated in the absence of puromycin. To
generate histone H2B-GFP cells, H1299, HCT116, and
U2OS cells were transfected with a plasmid expressing
both histone H2B-GFP and selected with blasticidine to
isolate single colonies. For NIH3T3, cells were infected
www.aacrjournals.org
Cell viability analysis and flow cytometry
Trypan blue analysis and flow cytometry after propidium iodide staining were carried out as described (25).
Live cell imaging and confocal microscopy
Cells were seeded onto poly-lysine–coated glass plates
and imaged using a TE2000E-PFS inverted fluorescent
microscope (Nikon) equipped with a SPOT BOOST
EMCCD camera (Diagnostic Instrument) and a INU-NIF1 temperature, humidity, and CO2 control system (Tokai
Hit). Data acquisition was carried out at 3 minutes/frame.
For confocal microscopy, cells grown on poly-L-lysine–
treated cover slips were fixed with 3% formaldehyde and
2% sucrose in PBS for 5 minutes. The cells were washed 3
times with PBS (all washes were 5 minutes each), permeabilized with 3% bovine serum albumin (BSA), and 0.4%
Triton X-100 in PBS for 30 minutes, and washed 3 times
with wash buffer (2% BSA and 0.2% Triton X-100 in PBS).
The cells were incubated with primary antibodies at 4 C
for 16 hours. After washing 5 times with wash buffer, the
cells were incubated with Alexa Fluor-488 goat antihuman IgG or Alexa Fluor-594 goat anti-mouse IgG secondary antibody (Invitrogen) for 1 hour. The cells were
washed 5 times with wash buffer and stained with
Hoechst 33258 (0.5 mg/mL in wash buffer) for 5 minutes.
After washing 3 times with wash buffer, the cover slips
were mounted with 2% N-propyl-gallate in glycerol.
Images were acquired with a Zeiss LSM510 confocal laser
scanning microscope (Carl Zeiss). Images were processed
with ImageJ software (NIH) from eight 0.38-mm z-stacks.
Antibodies and immunological methods
Monoclonal antibodies against b-actin (26), CDK1
(27), cyclin A2 (28), cyclin B1 (22), FLAG (29), a-tubulin
(30), and polyclonal antibodies against FLAG (21)
were obtained from sources as described previously.
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On et al.
Polyclonal antibodies against BUBR1 (Bethyl Laboratories), phospho-CDK1Tyr15 (Cell Signaling Technology), phospho-CHK1Ser317 (Abcam), CDC20, phosphohistone H3Ser10 (Santa Cruz Biotechnology), monoclonal
antibodies against p31comet (Abcam), CDC27, MAD2,
and cleaved PARP(Asp214; BD Biosciences), human
nuclear ANA-centromere CREST autoantibody (Fitzgerald Industries), CHK1, cyclin E2, and securin (Santa
Cruz Biotechnology) were obtained from the indicated
suppliers. Immunoblotting and immunoprecipitation
were carried out as described (24).
Results
Time-lapse microscopy reveals different cell fates
following the abrogation of the G2 DNA damage
checkpoint
HeLa cells were used to study cell fate after checkpoint
abrogation because they lack the p53-dependent G1 DNA
damage checkpoint. A subline that expressed both histone H2B-GFP and mRFP1-APC/C biosensor (containing
cyclin B1’s destruction box) was generated to allow the
simultaneous monitoring of DNA and APC/C activity,
respectively, in living cells. Figure 1A shows that irradiation with IR (15 Gy) triggered a G2-M arrest. Lower doses
were unable to trap all the cells in G2-M over the entire
course of the experiment (Supplementary Fig. S1 and data
not shown). Both caffeine and UCN-01 could uncouple
the checkpoint and promote progression into G1. Consistent with checkpoint bypass, histone H3Ser10 phosphorylation was strongly stimulated (Fig. 1B). As expected,
the increase in histone H3Ser10 phosphorylation and G1
population could be suppressed by inhibition of CDK1
with RO3306 (31). As a positive control, downregulation
of CHK1 with siRNA also promoted G1 entry (Fig. 1C)
and histone H3Ser10 phosphorylation (Fig. 1D) in
damaged cells.
To track the behavior of individual cells, they were
subjected to time-lapse microscopy. In contrast to control cells, a very small number of the irradiated cells
underwent mitosis over the 12-hour period of imaging
(Fig. 2A). They stayed in interphase and exhibited
low APC/C activity (Supplementary Video S1). These
data indicated that IR induced a G2 arrest without
causing significant cell death over the course of our
experiments.
Treatment of the irradiated cells with UCN-01 stimulated mitosis (Fig. 2A). Interestingly, a population of the
UCN-01–treated cells were able to degrade the APC/C
biosensor and undergo anaphase (Fig. 2B and Supplementary Video S2). Although 35% were killed during
mitosis, 50% of the cells were able to complete mitosis
successfully and progress into G1 (see Fig. 3D). The killed
cells were typically trapped in mitosis for a long period of
time without chromosomal segregation before undergoing apoptosis (Supplementary Video S3). In this study,
we defined these unscheduled mitotic cells that resulted
directly in apoptosis as undergoing mitotic catastrophe.
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Mol Cancer Ther; 10(5) May 2011
Treatment with UCN-01 alone did not affect survival
(Fig. 2A).
Although a subset of cells were able to complete mitosis after UCN-01 treatment, the duration of the mitosis
was considerably longer than that of the unperturbed
cycle (Fig. 2C). Most of the extension was due to a delay in
anaphase onset (Fig. 2D). For cells that underwent mitotic
catastrophe, the duration of mitosis was further extended
(Fig. 2A). We noted that cells that were able to enter G1
never spent longer than 4 hours in mitosis.
Collectively, these time-lapse analyses revealed that
while a population of cells was killed by mitotic catastrophe following checkpoint abrogation, a subset was
able to finish mitosis and enter G1.
The onset and extent of mitotic catastrophe are
influenced by the dose of radiation
We next examined whether the different fates after
checkpoint abrogation were influenced by the extent of
DNA damage. Cells were irradiated with either 15 or 40
Gy of IR before UCN-01 treatment. A G2-M arrest was
induced with both doses of IR (Fig. 3A and Supplementary Fig. S1). As shown above, treatment of 15 Gy–irradiated cells with UCN-01 prompted the appearance of G1
cells after 12 hours. Further incubation (24 hours) resulted
in an increase in apoptotic (sub-G1) cells. Tetraploids (8N)
were also induced, consistent with the fact that some cells
underwent abortive cytokinesis followed by genome
reduplication (see below). In contrast, cells treated with
IR (40 Gy) and UCN-01 continued to display 4N DNA
contents, with considerably more apoptotic cells and
fewer tetraploids.
The lack of G1 cells after IR (40 Gy) could be due to a
failure to enter mitosis even in the presence of UCN-01.
To exclude this possibility, the expression of various
mitotic markers was analyzed (Fig. 3B). Both histone
H3Ser10 and securin were strongly phosphorylated following UCN-01 treatment, indicating precocious mitosis.
Thus, the lack of 2N cells after IR (40 Gy) was not due an
inhibition of mitotic entry.
Time-lapse microscopy further confirmed that the
number of cells forced into mitosis by UCN-01 was
not affected by the level of DNA damage (Fig. 3C).
However, the onset of mitosis in 40 Gy–irradiated cells
was considerably delayed (average 300 minutes, compare to 230 minutes after 15 Gy). The delay in mitosis
after high dose of IR could be due to a more robust
activation of the DNA damage checkpoint and the
subsequent time required for shutting it down. As
expected, both doses of IR were able to activate
CHK1 (CHK1Ser317 phosphorylation) and induce
CDK1Tyr15 phosphorylation (Fig. 3B). However, CHK1
was activated more strongly in 40 Gy–irradiated cells.
Furthermore, CDK1Tyr15 phosphorylation appeared to
take longer to be reversed by UCN-01. The apparent
reduction of CHK1 signals at later time points was
probably due to the fuzziness of the bands caused by
mitotic phosphorylation shifts (32).
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Determinants of Mitotic Catastrophe
A
IR
IR + UCN-01
IR + caffeine
C
+RO3306
+IR
2N 4N
Cell
–IR
Cell
–RO3306
2N 4N
2N 4N
DNA
B
CHK1 siRNA
Control
2N 4N
2N 4N
DNA
D
IR:
+
+
+
+
+
CHK1 siRNA:
–
+
–
+
Caffeine:
UCN-01:
–
–
+
–
+
–
–
+
–
+
IR:
–
–
+
+
RO3306:
+
–
+
–
+
CHK1
H3(S10-p)
H3(S10-p)
CDK1(Y15-p)
CDK1(Y15-p)
Cyclin A2
Cyclin A2
PARP
B1
PARP (cleaved)
Cyclin B1
Actin
B1
CHK1
Cyclin B1
PARP
(cleaved)
1
2
3
4
5
Actin
1
2
3
4
Figure 1. Uncoupling of the G2 DNA damage checkpoint by UCN-01. A, UCN-01 can induce precocious cell cycle progression in damaged cells. HeLa
cells were irradiated and incubated for 16 hours. The cells were then treated with caffeine, UCN-01, and RO3306 as indicated. After 12 hours, the cells were
fixed and analyzed by flow cytometry. B, cells were treated as shown in A. Cell-free extracts were prepared and the expression of the indicated
proteins was detected by immunoblotting. Equal loading of lysates was confirmed by immunoblotting for actin. C, unscheduled mitotic entry after CHK1
depletion. HeLa cells were transfected with control or CHK1 siRNA. After 6 hours, the cells were irradiated, grown for 28 hours, and analyzed by flow cytometry.
D, cells were treated as shown in C. Lysates were prepared analyzed by immunoblotting.
Significantly, checkpoint-abrogated cells spent a longer
time in mitosis and underwent more mitotic catastrophe
following the higher dose of irradiation (compare Fig. 3C
with Fig. 2A; quantified in Fig. 3D). Collectively, these
data indicate that while a more severe level of DNA
damage did not curtail the ability of UCN-01 to induce
mitosis, a longer time was required for mitotic entry and
more cells underwent mitotic catastrophe.
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Cytokinesis failure is not a key determinant factor
of mitotic catastrophe
Although about half of checkpoint-abrogated cells
could complete mitosis and progress into G1, a substantial
portion was unable to complete cytokinesis properly (72
8% of mitotic cells, average and SD of 3 experiments;
Supplementary Video S4). This generated tetraploid G1
cells containing multiple nuclei. Cleavage furrow
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A
IR
Control
IR + UCN-01
UCN-01
Mitosis
(78%)
Cell
Cell
Mitosis
(82%)
Cell
Cell
MC
(24%)
Mitosis
(57%)
120 240 360 480 600 720
Time (min)
B
120 240 360 480 600 720
Time (min)
0
APC/C
120 240 360 480 600 720
Time (min)
0
120 240 360 480 600 720
Time (min)
D
C
H2B
Length of mitosis (min)
Bright field
0
120
120
100
100
80
80
Time (min)
0
60
40
20
Metaphase–
anaphase
Prometaphase–
metaphase
60
40
20
0
Control
IR
IR + UCN-01
0
Control IR + UCN-01
Figure 2. Various cell fates are induced by UCN-01. A, mitotic catastrophe and survival after UCN-01 challenge. HeLa cells expressing histone H2B-GFP
and mRFP1-APC/C biosensor were untreated (n ¼ 62), irradiated and grown for 16 hours (n ¼ 66), UCN-01–treated (n ¼ 48), or irradiated (16 hours)
followed by UCN-01 treatment (n ¼ 62). The cells were then subjected to time-lapse microscopy to track individual cells for 12 hours. Gray, interphase; black,
mitosis (from DNA condensation to anaphase) or mitotic catastrophe; truncated bars, cell death. The time of cell death after mitosis is defined by the death of
one of the daughters. The percentages of cells that completed mitosis or underwent mitotic catastrophe (MC) are shown [a portion of cells that were
still trapped in an extremely long mitosis at the end of the 12-hour imaging was also categorized as MC—longer imaging revealed that they subsequently also
underwent apoptosis (data not shown)]. B, Representative still images from time-lapse microscopy of IR- and UCN-01–treated cells. Cells were treated
and imaged as shown in A. An example of a cell that could complete mitosis is shown. The full video can be found in Supplementary data (Supplementary Video
S2). C, mitosis is extended in UCN-01–treated cells. Cells were treated and imaged as shown in A. The duration of mitosis in control (n ¼ 50), cells treated with
IR (n ¼ 7, only a very small number of cells could enter mitosis), or IR followed by UCN-01 (n ¼ 35) are shown (mean 90% CI). D, anaphase is delayed in UCN01–induced mitosis. Control (n ¼ 50) or IR- and UCN-01–treated cells (n ¼ 35) were analyzed by time-lapse microscopy as shown in A. The time of
prometaphase–metaphase and metaphase–anaphase was quantified (mean 90% CI).
regression and abortive cytokinesis were likely to be
caused by lagging chromosomes or chromosomal fragments (DNA with and without kinetochore staining,
respectively), which were found in more than 50% of
unscheduled mitosis (Supplementary Fig. S2A). Although
some researchers include cytokinesis failure as the definition of mitotic catastrophe, there is no consensus in the
field. We have restricted our definition of mitotic catastrophe to inappropriate mitosis that results directly in
apoptosis (11).
To address whether cytokinesis failure per se plays a
role in cell death, cytokinesis was completely abolished
by inhibiting Aurora B with ZM-447439. Supplementary
Figure S2B shows that UCN-01–mediated mitotic catastrophe was not increased by ZM-447439 (quantified in
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Mol Cancer Ther; 10(5) May 2011
Supplementary Fig. S2D). A caveat is that inhibition of
Aurora B also abolished anaphase and shortened mitosis
(Supplementary Video S5). Therefore, the actin inhibitor
DCB was used as an alternative method to inhibit cytokinesis (Supplementary Video S6). Supplementary Figure
S2C indicates that mitotic catastrophe was not affected by
DCB (quantified in Supplementary Fig. S2D), suggesting
that cytokinesis failure is not a major determining factor
of mitotic catastrophe.
Mitotic catastrophe is suppressed by weakening of
the spindle-assembly checkpoint
Given that a portion of the checkpoint-bypassed cells
could complete mitosis and enter the next G1 phase, we
next sought to define mitotic factors that influence mitotic
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Determinants of Mitotic Catastrophe
a cell line that expressed FLAG-tagged p31comet under
the control of doxycycline. Supplementary Figure S3A
confirms that recombinant p31comet could bind to
catastrophe. To this end, the spindle-assembly checkpoint was subdued by overexpression of p31comet, a
potent inhibitor of MAD2 (33, 34). First, we generated
A
B
IR
UCN-01
–
–
–
+
15 Gy
–
Time = 12 h
Time = 24 h
IR (Gy):
UCN-01/NOC:
Time (h):
– – – – 15 15 15 15 15 15 40 40 40 40 40 40
– + + + – + – + – + – + – + – +
6 6 12 24 6 6 12 12 24 24 6 6 12 12 24 24
H3 (S10-p)
Cyclin B1
Securin
CDK1 (Y15-p)
CDK1
CHK1 (S317-p)
15 Gy
+
CHK1
Cyclin E2
40 Gy
–
PARP (cleaved)
Actin
+
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Cell
40 Gy
2N 4N
2N 4N
8N
DNA
C
D
IR(40 Gy) + UCN-01
IR(40 Gy)
100
15 Gy IR + UCN-01
40 Gy IR + UCN-01
Cell
Cell
MC
(84%)
Cell (%)
75
50
25
Mitosis
(14%)
0
120 240 360 480 600 720
Time (min)
0
0
Mitosis
Mitotic
Interphase
catastrophe
120 240 360 480 600 720
Time (min)
Figure 3. Both the onset and extent of mitotic catastrophe are dependent on the level of DNA damage. A, high dose of IR prohibits UCN-01–induced G1
entry. HeLa cells were irradiated (15 or 40 Gy), grown for 16 hours, and treated with UCN-01 as indicated. At 12 and 24 hours after UCN-01 addition, DNA
contents were analyzed by flow cytometry. B, dose-dependent activation of CHK1. HeLa cells were irradiated (15 or 40 Gy), grown for 16 hours, and
treated with UCN-01 as indicated. Nocodazole was included to trap checkpoint-bypassed cells in mitosis. At the indicated time points after UCN-01 addition,
the cells were harvested and analyzed by immunoblotting. C, Delayed mitotic entry and massive mitotic catastrophe after IR (40 Gy) and UCN-01 treatment.
Cells were treated with IR (40 Gy) followed by UCN-01 as shown in A. Time-lapse microscopy was used to track individual cells for 12 hours. Key and
definition are the same as given in Fig. 2A (n ¼ 50). D, massive mitotic catastrophe in cells treated with IR (40 Gy) and UCN-01. Cells were treated with IR (40
Gy), UCN-01, and analyzed as shown in A (n ¼ 50). Cells that completed mitosis without dying (mitosis), die during mitosis (mitotic catastrophe), or did not enter
mitosis throughout the imaging period (interphase) were quantified (mean SD of 3 experiments).
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On et al.
endogenous MAD2. Mitotic block induced by nocodazole
could be overcome by p31comet, thereby resulting in
genome reduplication (Supplementary Fig. S3B). This
validated that p31comet overexpression was able to disrupt the MAD2-dependent spindle-assembly checkpoint.
Figure 4A shows that the unscheduled histone H3Ser10
phosphorylation triggered by UCN-01 in irradiated cells
was abolished in the presence of p31comet. Likewise, cell
death after checkpoint abrogation was also inhibited by
p31comet (Fig. 4A). Using time-lapse microscopy, the
striking differences between control and p31comet-expressing cells were revealed more directly (Fig. 4C). Mitotic
A
catastrophe was reduced from 37% to nil after p31comet
was switched on.
As an alternative way to suppress the spindle-assembly checkpoint, we also used a previously characterized
shRNA against MAD2 (20, 22). Similar to p31comet overexpression, MAD2 depletion was sufficient to reverse
the mitotic catastrophe triggered by IR and UCN-01
(Fig. 4C).
To verify that the effects of the shRNA were specific
to MAD2, we used a functional rescue system that
expressed both MAD2 shRNA and cDNA (containing
silence mutations that rendered it resistant to the
B
p31comet :
IR:
UCN-01:
– + – + – + – +
– – – – + + + +
– – + + – – + +
18
p31
14
Cell death (%)
FLAG
CDK1(Y15-p)
Cyclin B1
– p31
+ p31
16
12
10
8
6
4
H3(S10-p)
2
0
Actin
UCN-01
1
C
IR
IR + UCN-01
2 3 4 5 6 7 8
p31comet + IR + UCN-01
IR + UCN-01
MAD2 shRNA + IR + UCN-01
Cell
Cell
Cell
MC
(46%)
Mitosis
(67%)
Mitosis
(66%)
Mitosis
(44%)
0
120 240 360 480 600 720
Time (min)
0
120 240 360 480 600 720
Time (min)
0
120 240 360 480 600 720
Time (min)
Figure 4. Expression of p31comet attenuates UCN-01–mediated mitotic catastrophe. A, mitosis induced by checkpoint bypass is abolished by p31comet. FLAGp31comet/HeLa cells were grown in the absence or presence of doxycycline for 30 hours to turn on or off the expression of FLAG-p31comet, respectively. They
were treated with IR (16 hours) followed by UCN-01 (12 hours) as indicated. Lysates were prepared and analyzed by immunoblotting. B, p31comet suppresses
UCN-01–mediated cell death. Cells were treated as shown in A. Cell viability was determined with trypan blue exclusion analysis (mean SD of 3
experiments). C, expression of MAD2 shRNA or p31comet suppresses mitotic catastrophe. HeLa cells expressing histone H2B-GFP were transfected with
control vector (n ¼ 50), plasmids expressing FLAG-p31comet (n ¼ 52), or MAD2 shRNA (n ¼ 74) together with a YFP marker. The cells were irradiated,
grown for 16 hours, before treated with UCN-01. Time-lapse microscopy was used to track individual cells for 12 hours. Key and definition are the same as
given in Fig. 2A.
790
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Determinants of Mitotic Catastrophe
shRNA; ref. 20). Supplementary Figure S4A shows that
while mitotic catastrophe was abolished by MAD2
shRNA, it was restored by coexpression of MAD2.
The shortening of mitosis by MAD2 shRNA was also
reversed by MAD2 (Supplementary Fig. S4B). These
experiments verified the specificity of MAD2 shRNA
on mitotic catastrophe.
Not surprisingly, UCN-01–induced mitosis was shortened considerably in cells expressing p31comet or MAD2
shRNA (Supplementary Fig. S4C). Although these cells
could undergo mitosis without dying, neither metaphase
plate formation nor cytokinesis occurred properly. Consequently, the nuclei of the daughter cells were highly
fragmented (Supplementary Videos S7 and S8).
Finally, we found that expression of p31comet could
even rescue the massive mitotic catastrophe induced by
IR (40 Gy) and UCN-01 (Supplementary Fig. S5, compare
Fig. 3C). Taken together, these results indicate that weakening of the spindle-assembly checkpoint reduced mitotic catastrophe.
Mitotic catastrophe can be promoted by delaying
mitotic exit
Given that mitotic catastrophe can be suppressed by
weakening of the spindle-assembly checkpoint, we next
tested the converse conditions of delaying mitotic exit.
Figure 5A shows that both CDC20 and p31comet could be
effectively depleted with siRNAs. As expected, phosphorylation of checkpoint components including BUBR1
and CDC27 was enhanced. Moreover, depletion of
CDC20 or p31comet lengthened mitosis considerably (Supplementary Fig. S6A). This also enhanced IR and UCN01–induced cell death as measured by trypan blue exclusion assays (Supplementary Fig. S6B). Consistent with
these observations, fewer G1 cells were found after checkpoint abrogation in CDC20-depleted cells (Fig. 5B).
Tracking of individual cells confirmed that mitotic catastrophe was significantly enhanced after depletion of
CDC20 and p31comet (Fig. 5C), with cells spending a long
period of time in metaphase before dying (Supplementary Videos S9 and S10). As a control, the siRNAs alone
(without IR/UCN-01) extended mitosis but did not
induce cell death (data not shown). Collectively, these
data indicate that mitotic catastrophe could be enhanced
by delaying the activation of APC/C.
Different cell types display different susceptibility
to mitotic catastrophe
A prediction from the above data is that cells with
different intrinsic mitotic time may display different
susceptibility to mitotic catastrophe. Therefore, we next
examined the responses of several commonly used cell
lines to IR and UCN-01. Histone H2B-GFP was first
stably expressed in these cells; and the same conditions
of checkpoint abrogation and live cell imaging were
repeated. Figure 6A shows that HeLa exhibited the
longest unperturbed mitosis among the cell lines. Mitosis was inhibited by both doses of IR in all the cell lines
www.aacrjournals.org
(Supplementary Fig. S7). As expected, UCN-01 stimulated mitotic entry in the damaged cells. Unlike HeLa
cells, mitotic catastrophe could hardly be detected in
other cells (Fig. 6C and Supplementary Fig. S7). The
extension of the duration of mitosis after checkpoint
abrogation in other cell lines was also less than that in
HeLa cells (Fig. 6B). Nevertheless, H1299, which underwent mitotic catastrophe at a level second to HeLa, also
exhibited a relatively long mitosis after checkpoint
abrogation.
To ensure that the failure of the cell lines to undergo
mitotic catastrophe was not due to defects in apoptotic
pathways, we delayed the mitotic exit of U2OS cells by
depleting CDC20 as above. Transfection of CDC20
siRNA before IR and UCN-01 treatments significantly
increased the mitotic length and cell death (Fig. 6C and
Supplementary Fig. S8). No significant cell death was
induced by CDC20 depletion alone in U2OS cells (data
not shown). These results indicate that although the
ability of UCN-01 to induce mitotic catastrophe varies
greatly between different cell lines and appear to correlate with the intrinsic mitotic length, mitotic catastrophe could similarly be induced by extending the
duration of mitosis.
Discussion
In this study, time-lapse microscopy was used to carefully track the fate of individual cells after checkpoint
abrogation. We found that while mitosis was abolished
after HeLa cells were damaged with IR, addition of UCN01 was able to induce mitotic entry in more than 80% of
the population (Figs. 2A and 3D).
About half of the UCN-01–induced mitotic cells completed mitosis without undergoing mitotic catastrophe.
However, it is likely that the majority will not survive
during subsequent stages of the cell cycle. In fact, further
imaging (up to 48 hours) revealed extensive postmitotic
cell death (unpublished data). Cytotoxicity measured in
most of the previous studies on checkpoint abrogation is
a combination of cell death during mitosis and the subsequent cycles.
Conceptually, it is not immediately obvious why the
unscheduled mitosis should be a period of cytotoxicity.
DNA damage per se did not cause the cytotoxicity,
because irradiated HeLa cells remained viable over the
same period in G2 (Figs. 2A and 3C). We think that an
extensive trap in mitosis may contribute to the cell death.
Mitosis was generally lengthened following checkpoint
abrogation (Fig. 2A and C), caused mainly by a delay of
anaphase onset (Fig. 2D).
The duration of unperturbed mitosis in HeLa cells
was 45 minutes (Fig. 2C). After IR (15 Gy) and UCN-01
treatment, cells that could complete mitosis displayed
an average mitotic length of 100 minutes (Fig. 2C). We
observed that mitosis lasting more than 4 hours resulted
in cell death. Hence, whether a cell underwent mitotic
catastrophe may depend on the length of time spent in
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On et al.
A
B
Control
CDC20 siRNA
CDC20 siRNA: – + –
p31comet siRNA: – – +
Control
CDC20
p31comet
UCN-01
BUBR1
Cyclin B1
IR
CDC27
Actin
3
IR + UCN-01
Cell
1 2
2N 4N
2N 4N
DNA
C
p31comet siRNA + IR + UCN-01
CDC20 siRNA + IR + UCN-01
IR + UCN-01
MC
(42%)
MC
(76%)
Cell
Cell
Cell
MC
(65%)
Mitosis
(54%)
Mitosis
(28%)
Mitosis
(6%)
0
120 240 360 480 600 720
Time (min)
0
120 240 360 480 600 720
Time (min)
0
120 240 360 480 600 720
Time (min)
Figure 5. Mitotic catastrophe can be promoted by delaying mitotic exit. A, downregulation of CDC20 and p31comet. HeLa cells were transfected with
control or siRNAs against CDC20 or p31comet. Lysates were prepared and analyzed by immunoblotting (asterisks, phosphorylated forms of BUBR1 and
CDC27). B, depletion of CDC20 inhibits mitotic exit. HeLa cells were transfected with either control or CDC20 siRNA. After 6 hours, the cells were irradiated,
grown for 16 hours, and treated with UCN-01. After 12 hours, DNA contents were analyzed with flow cytometry. C, downregulation of CDC20 or p31comet
enhances mitotic catastrophe. HeLa cells expressing histone H2B-GFP and mRFP1-APC/C biosensor were transfected with control siRNA (n ¼ 50),
CDC20 siRNA (n ¼ 68), or p31comet siRNA (n ¼ 74). The cells were irradiated, grown for 16 hours, and treated with UCN-01. Individual cells were tracked with
time-lapse microscopy for 12 hours. Key and definition are the same as given in Fig. 2A.
mitosis. In support of this hypothesis, weakening of the
spindle-assembly checkpoint by p31comet overexpression or MAD2 depletion suppressed mitotic catastrophe
(Fig. 4). Conversely, lengthening of mitosis by depletion
of CDC20 or p31comet increased mitotic catastrophe
(Fig. 5 and Supplementary Fig. S6). These results are
in agreement with the findings that the spindle-assembly checkpoint is important for mitotic catastrophe
induced by topoisomerase II inhibitors followed by
UCN-01 treatment (8, 35), or for cell death in cases
792
Mol Cancer Ther; 10(5) May 2011
where the DNA damage checkpoint has been weakened
(36).
Irradiation of HeLa cells with a higher dose of IR (40
Gy) did not prevent UCN-01–mediated mitosis. But the
time between UCN-01 treatment and mitotic entry was
increased (Fig. 3C). This dose-dependent delay in mitotic
entry was reproducible in different cell lines (Supplementary Fig. S7). We attribute this mainly to a more
robust G2 DNA damage checkpoint that required a longer
time to be shut down (Fig. 3B). Significantly, more cells
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Determinants of Mitotic Catastrophe
A
Mitotic duration (min)
50
40
30
20
10
0
HeLa NIH3T3 H1299 HCT116 U2OS
B
200
Mitotic duration (min)
175
150
125
100
75
50
25
0
HeLa NIH3T3 H1299 HCT116 U2OS
C
Mitotic catastrophe (%)
100
15 Gy
40 Gy
75
50
25
0
HeLa NIH3T3 H1299 HCT116 U2OS
CDC20 siRNA: –
–
–
–
–
U2OS
+
Figure 6. Different cell lines display different susceptibility to mitotic
catastrophe. A, the unperturbed mitotic length of different cell lines. The
indicated cell lines, which expressed histone H2B-GFP, were subjected to
time-lapse microscopy. Mitosis length in individual cells was analyzed (n ¼
50, mean 90% CI). B, mitotic length after checkpoint abrogation. The
indicated histone H2B-GFP-expressing cell lines were irradiated (15 Gy),
grown for 16 or 24 hours (for U2OS and NIH3T3), and treated with UCN-01.
Individual cells were tracked with time-lapse microscopy (n ¼ 50) to
analyze the duration of mitosis (mean 90% CI). C, different cell lines
exhibit different susceptibility to mitotic catastrophe. The indicated histone
H2B-GFP–expressing cell lines were transfected with control or CDC20
siRNA. The cells were irradiated with either 15 or 40 Gy, grown for 16 or 24
hours (for U2OS and NIH3T3), and treated with UCN-01. Mitotic
catastrophe was analyzed by time-lapse microscopy (12 hours, n ¼ 50).
underwent mitotic catastrophe after 40 Gy of IR (Fig. 3D).
We believe that this was in part due to a longer mitosis:
the average mitotic length (for cells that completed mito-
www.aacrjournals.org
sis successfully) was 100 45 minutes (15 Gy) and 122 45 minutes (40 Gy; n ¼ 50). Indeed, the massive mitotic
catastrophe induced in 40 Gy–irradiated cells was
reversed when mitosis was shortened with p31comet (Supplementary Fig. S5).
Several other cell lines are more resistant to mitotic
catastrophe than HeLa cells under the same conditions
(Fig. 6C). Interestingly, these cell lines displayed a shorter
mitosis than HeLa (Fig. 6A and B). Whether this is the
explanation of their relative resistance to mitotic catastrophe remained to be deciphered. What is clear is that
prolonging mitosis by CDC20 siRNA also increased
mitotic catastrophe in U2OS cells (Fig. 6C). The status
of p53 does not appear to have a significant effect, as both
p53-positive (U2OS and HCT116) and negative (HeLa,
H1299, and NIH3T3) cells were examined.
What could be the link between prolonged mitosis and
cell death? Cyclin B–CDK1 activity is known to be associated with cell death. For example, mitotic catastrophe
can be promoted by cyclin B1–CDK1 overexpression (37,
38) and inhibited by cyclin B1 knockdown (39). Although
the underlying principles of CDK1-mediated toxicity
remain largely unresolved, a few targets that affect survival, including members of the BCL-2 family have been
identified (11).
In cells that contain defective DNA damage checkpoints, mitotic catastrophe represents a mechanism that
prevent further genome instability. Our data suggest
that additional defects in the spindle-assembly checkpoint may undermine the protective role of mitotic
catastrophe. Our data also indicate that the status of
the checkpoint is critical in determining whether cancer
cells are eliminated by treatments with IR and UCN-01.
Drugs that lead to activation of the checkpoint (e.g.,
spindle poisons) may be used in conjunction with UCN01 to trigger more effective mitotic catastrophe. Importantly, our results indicate that extending mitosis by
downregulation of CDC20 or p31comet has the same
effects on mitotic catastrophe as using a higher dose
of IR. An implication of this is that a lower dose of IR can
be used (thus causing less general toxicity) if UCN-01 is
administered together with a drug that delays mitotic
exit.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Grant Support
This work was supported in part by the Research Grants Council
grants 662208 and CA06/07.sc02 to R.Y.C. Poon.
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 18 U.S.C. Section 1734 solely to indicate
this fact.
Received August 27, 2010; revised February 16, 2011; accepted March 4,
2011; published OnlineFirst March 23, 2011.
Mol Cancer Ther; 10(5) May 2011
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793
Published OnlineFirst March 23, 2011; DOI: 10.1158/1535-7163.MCT-10-0809
On et al.
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Molecular Cancer Therapeutics
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Published OnlineFirst March 23, 2011; DOI: 10.1158/1535-7163.MCT-10-0809
Determinants of Mitotic Catastrophe on Abrogation of the G2
DNA Damage Checkpoint by UCN-01
Kin Fan On, Yue Chen, Hoi Tang Ma, et al.
Mol Cancer Ther 2011;10:784-794. Published OnlineFirst March 23, 2011.
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