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Mol Cell Biol, February 1998, p. 1055-1064, Vol. 18, No. 2
Center for Cancer
Research,1
Department of
Biology,2 and
Howard Hughes Medical
Institute,3 Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139
Received 5 September 1997/Returned for modification 9 October
1997/Accepted 10 November 1997
The p53 tumor suppressor gene product is known to act as part of a
cell cycle checkpoint in G1 following DNA damage. In order to investigate a proposed novel role for p53 as a checkpoint at mitosis
following disruption of the mitotic spindle, we have used time-lapse
videomicroscopy to show that both p53+/+ and
p53 The proper execution of events in
the eukaryotic cell cycle is regulated by a number of different
checkpoints. For example, to ensure stable maintenance of the genome,
cells arrest in G1 or G2 upon detection of DNA
damage, providing time for DNA repair before the initiation of DNA
synthesis or entry into mitosis (11). Other checkpoints
function during mitosis to monitor successful assembly of the spindle
and control the initiation of metaphase, thus protecting the cell from
chromosome missegregation (37). Genes that are required for
cellular arrest at different checkpoints have been identified,
demonstrating that each checkpoint is a genetic pathway activated by
specific signals. The inactivation of checkpoint genes leads to
increased mutation rate, chromosome loss, or changes in ploidy,
depending on the genes affected (34). Interestingly, some
genes that are mutated in human cancers are involved in checkpoint
functions, suggesting that without such controls in place, the
resulting genetic damage predisposes cells to malignancy
(38).
The p53 tumor suppressor gene is mutated in over half of all sporadic
human cancers. p53 has an essential role in the G1
checkpoint in response to DNA-damaging agents such as radiation
(20, 21, 23). Functional analysis of the p53 protein has
shown that it is a transcription factor with sequence-specific DNA
binding activity (12, 22, 42). After DNA damage, p53
activates the transcription of several downstream target genes,
including p21, an inhibitor of cyclin-dependent kinases (CDKs)
(10). The induction of p21 causes subsequent arrest in the
G1 phase of the cell cycle by binding of cyclin-CDK
complexes (14, 15, 41). Additional p53 target genes are
likely to cooperate with p21 in implementing G1 arrest, as
p21-deficient mouse cells are only partially defective in their DNA
damage arrest response, while p53-deficient cells are completely
defective (2, 7).
Recently, p53 has been proposed to have an additional, novel function
as a checkpoint at mitosis. Wild-type fibroblasts arrest when mitotic
spindle assembly is disrupted by the addition of drugs which bind
microtubules. However, studies have shown that p53-deficient
fibroblasts fail to arrest under such conditions but instead undergo a
new round of DNA synthesis in the absence of cell division, becoming
polyploid (5, 8, 33). This phenotype is very similar to that
observed in yeast strains that have inactivated spindle assembly
checkpoints. Saccharomyces cerevisiae strains with mutations
in the MAD or BUB genes, which monitor spindle assembly, become
polyploid when treated with spindle inhibitors (17, 27).
Based on this similarity, one might conclude that, like the MAD and BUB
gene products, p53 monitors spindle integrity and can thus be termed a
mitotic checkpoint. Also, additional data have suggested that the
spindle components themselves may be under p53 regulation, as p53 has
been shown to localize to the centrosome (1) and
p53 In this study, we have characterized in greater detail the
p53-dependent checkpoint following disruption of the mitotic spindle with microtubule-destabilizing drugs. Upon examining the response of
wild-type and p53-deficient mouse embryo fibroblasts to spindle inhibitors, we observed that cells of either genotype underwent a
transient arrest at mitosis and subsequently progressed into a
G1-like state without ever completing cell division.
Wild-type MEFs remained arrested in this state, while
p53 Cells and cell culture.
p53+/+ and
p53
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Characterization of the p53-Dependent Postmitotic
Checkpoint following Spindle Disruption
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
/ murine fibroblasts treated with the spindle drug
nocodazole undergo transient arrest at mitosis for the same length of
time. Thus, p53 does not participate in checkpoint function at mitosis.
However, p53 does play a critical role in nocodazole-treated cells
which have exited mitotic arrest without undergoing cytokinesis and have thereby adapted. We have determined that in nocodazole-treated, adapted cells, p53 is required during a specific time window to prevent
cells from reentering the cell cycle and initiating another round of
DNA synthesis. Despite having 4N DNA content, adapted cells are similar
to G1 cells in that they have upregulated cyclin E
expression and hypophosphorylated Rb protein. The mechanism of the
p53-dependent arrest in nocodazole-treated adapted cells requires the
cyclin-dependent kinase inhibitor p21, as p21/
fibroblasts fail to arrest in response to nocodazole treatment and
become polyploid. Moreover, p21 is required to a similar extent to
maintain cell cycle arrest after either nocodazole treatment or
irradiation. Thus, the p53-dependent checkpoint following spindle disruption functionally overlaps with the p53-dependent checkpoint following DNA damage.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
/ mouse embryonic fibroblasts (MEFs) contain
abnormal numbers of centrosomes (13). However, later work
has shown that in cells treated with spindle inhibitors, p53 is neither
expressed during mitosis nor required for mitotic arrest
(33). Instead, expression of p53 protein occurs only after
cells exit mitotic arrest and progress to an interphase state while
retaining 4N DNA content. These data argue that p53 does not function
as a mitotic checkpoint but acts at some subsequent point in the cell
cycle to induce arrest following a defect in M phase.
/ MEFs initiated another round of DNA synthesis.
Using time-lapse videomicroscopy, we were able to define the length of
mitotic arrest and establish precisely the timing of S phase reentry in p53/ MEFs after exit from mitotic arrest. We also
demonstrated a requirement for the p53 target gene product, p21, in
executing cell cycle arrest after spindle disruption. Upon further
characterization of arrested cells, we determined that cells expressed
molecular markers associated with the G1 phase of the cell
cycle, despite having 4N DNA content. These data demonstrate that the
p53-dependent checkpoint in cells with disrupted mitotic spindles has
strong similarity to the p53-dependent checkpoint in G1
following DNA damage. Our data suggest that rather than having a novel
role as a spindle checkpoint, p53 functions in the G1 phase
of the cell cycle and via the same downstream pathway in murine cells which have sustained either DNA damage or microtubule disruption.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
/ MEFs were derived from 13.5-day-old embryos and
used between passages 3 and 8 (18). MEFs were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum supplemented with penicillin and streptomycin and grown in
an atmosphere containing 5% CO2. For flow cytometry and immunoblot experiments, MEFs were plated at a density of 5 × 105 to 1 × 106 cells per 100-mm-diameter
dish 24 h in advance of experiments. Nocodazole (Sigma) was kept
as a 1 mg/ml stock in dimethylsulfoxide. Nocodazole treatment of MEFs
was performed at a concentration of 0.125 µg of nocodazole/ml of
medium.
Flow cytometry.
Approximately 106 cells per
100-mm-diameter dish were detached in 0.25% trypsin and washed in
phosphate-buffered saline (PBS). Following centrifugation, cells were
resuspended in 0.5 ml of cold PBS. Cells were fixed by adding 4.5 ml of
cold 100% ethanol dropwise to the sample while vortexing gently and
then placing at 
20°C for at least 8 h. After fixation, cells
were pelleted out of ethanol, washed once with PBS, and resuspended in
20 µg of propidium iodide (Sigma) plus 200 µg of RNase (Sigma)/ml
of PBS. Cells were incubated at 37°C for 30 min and then allowed to
stain for at least 8 h at 4°C. Samples were analyzed for DNA content on a Becton Dickinson FACScan.
Immunofluorescence. A total of 105 MEFs were plated onto glass coverslips in a 34-mm-diameter tissue culture well. MEFs were allowed to adhere to coverslips for 24 h and were then changed into medium with or without nocodazole (0.125 µg/ml) and incubated for 24 h. Bromodeoxyuridine (BrdU) and fluorodeoxyuridine (FdU) (Sigma) were added to the medium from a 1,000× stock solution in H2O, for a final concentration of 3 µg of BrdU plus 0.3 µg of FdU/ml of medium; cells were incubated for an additional 4 h. Coverslips were then fixed in 4% paraformaldehyde-PBS for 20 min at room temperature, washed in PBS, permeabilized for 15 min in 0.25% Triton X-100-PBS, and washed again with PBS. For BrdU detection, slips were denatured in 1.5 N HCl for 10 min and washed several times with PBS. Slips were incubated with a murine monoclonal antibody to BrdU (1:50 dilution; Becton Dickinson) in 10% goat serum-PBS for 30 min at 37°C. After two washes in PBS, fluorescein-conjugated anti-mouse antibody (1:200 dilution; Jackson Immunoresearch Laboratories) was added in 10% goat serum-PBS and the slips were incubated for 30 min at 37°C. Slips were washed with PBS and stained for 5 min in 0.2 µg of 4',6-diamidino-2-phenylindole (DAPI; Sigma)/ml of PBS. Coverslips were mounted on glass slides with Mowiol and analyzed via fluorescence microscopy.
Time-lapse videomicroscopy. MEFs were grown on glass coverslips as for immunofluorescence. A single coverslip was placed in a dish containing 2 ml of HEPES-buffered DMEM plus 10% fetal bovine serum, with or without nocodazole (0.125 µg/ml). Each coverslip was observed for up to 24 h under a Nikon microscope. Recording was done by taking one picture frame every 8 s with a GYYR TLC2100 time-lapse videocassette recorder. During recording, MEFs were maintained at 37°C with a Delta temperature controller and kept in an environment of 5% CO2 by bubbling gas across the surface of the media. Pictures were printed out with a Sony color video printer. All equipment was obtained from Micro Video Instruments, Inc.
For time-lapse videomicroscopy followed by immunofluorescence, MEFs were grown on gridded coverslips (Bellco) placed in 34-mm-diameter wells, with 105 MEFs plated per well. A single square of the grid was followed by time-lapse videomicroscopy for 18 to 24 h. For BrdU incorporation assays, 2 µl of BrdU-FdU 1,000× stock solution was added directly to the 2 ml of media in the dish 4 h before the end of the experiment, for a final concentration of 3 µg of BrdU plus 0.3 µg of FdU/ml of medium. After recording was complete, the gridded slip was fixed in 4% paraformaldehyde-PBS and immunofluorescence was performed. The cells which had been recorded on video were identified during immunofluorescence analysis by locating the same square of the grid.Immunoblotting.
Whole-cell extracts were made by scraping
approximately 106 cells in 200 µl of boiling lysis buffer
(100 mM NaCl, 10 mM Tris [pH 8.0], 1% sodium dodecyl sulfate
[SDS]). Lysates were heated at 100°C for 10 min, quick frozen in a
dry ice-ethanol bath, and stored at 80°C. Protein concentration of
lysates was determined with a bicinchoninic acid kit (Pierce). For
cyclin E, cyclin B1, and p21 immunoblots, 100 µg of protein was
loaded per lane on an SDS-10% polyacrylamide gel (29:1). Gels were
electrophoresed at 150 V for 4 h and then transferred to
Immobilon-P membrane (Millipore) in low-molecular-weight transfer
buffer (25 mM Tris, 190 mM glycine, 20% methanol) for 12 h at 25 V at
4°C. Blots were blocked in 0.01% Tween 20-PBS (PBS-T) plus 5% dried
milk for 1 h at room temperature and then probed with primary
antibody diluted in block solution for 1 h at room temperature.
Antibodies and dilutions were as follows: cyclin E (Santa Cruz M-20),
1:200 dilution; cyclin B1 (Pharmingen GNS-1), 1:200 dilution; and p21
(Santa Cruz C19-G), 1:100 dilution. Blots were then washed with PBS-T
three times for 10 min. For p21 detection, blots were put through an additional incubation step in mouse anti-goat antibody (1:12,000; Jackson Immunoresearch Laboratories), diluted in block solution for
1 h at room temperature, and washed three times in PBS-T. All
blots were then incubated in peroxidase-linked secondary antibody (1:5,000 dilution; Amersham) for 1 h at room temperature and
washed three times in PBS-T. Blots were developed with chemiluminescent reagents and exposed to Kodak X-Omat 5 film for 1 to 20 min.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Because previous results regarding p53 function at mitosis were
contradictory, we chose to characterize in greater detail the effect of
p53 deficiency on cells treated with spindle inhibitors. To confirm a
requirement for p53 following spindle disruption, wild-type and
p53-deficient MEFs were grown in the presence of the
microtubule-destabilizing drug nocodazole. After 24 h of
treatment, cells were fixed and stained with propidium iodide for
analysis by flow cytometry (Fig. 1A).
Wild-type MEFs treated with nocodazole arrested with primarily a 4N DNA
content. A small subpopulation of wild-type cells that had 8N DNA
content was observed, perhaps reflecting the presence of cycling
tetraploid cells in the normal population, which would be predicted to
arrest with 8N DNA content upon spindle disruption. As described
previously, p53/ fibroblasts did not arrest with a 4N
DNA content upon drug treatment but continued through an additional
round of S phase to become 8N (Fig. 1A) (5, 8, 33).
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-BrdU) and nuclear
staining (DAPI). Immunofluorescence was performed on two different
wild-type and p53In order to examine specifically the fraction of cells capable of S
phase reentry following nocodazole treatment, cells were treated with
nocodazole for 24 h, pulsed with BrdU for an additional 4 h
while still in the presence of drug, and analyzed by immunofluorescence for anti-BrdU staining (Fig. 1B). Untreated wild-type and
p53/ MEFs had similar proportions of cells in S phase.
After 28 h of nocodazole treatment, very few BrdU-positive cells
were observed in the wild-type MEF population, consistent with their
ability to induce cell cycle arrest after nocodazole treatment.
However, many p53/ MEFs incorporated BrdU while in the
presence of nocodazole, indicating that they were able to reenter the
cell cycle and initiate DNA synthesis, despite having failed to undergo
proper mitosis. Both wild-type and p53/ cells were
observed to contain micronuclei following nocodazole treatment,
resulting from chromosome decondensation and subsequent reformation of
the nuclear membrane (24). These results confirm previous
observations (5, 8, 33) describing a p53-dependent arrest
following destabilization of the mitotic spindle.
One interpretation of the results described in Fig. 1 is that p53
functions in a checkpoint at mitosis, based on the fact that
p53/ MEFs behave abnormally when the mitotic spindle is
disrupted. To test this hypothesis directly, we used time-lapse
videomicroscopy to observe p53+/+ and p53/
MEFs during mitosis. If p53 were part of a mitotic checkpoint, then we
would expect to see a difference in the behavior of nocodazole-treated p53+/+ and p53/ MEFs at mitosis. Figure
2A shows a representative untreated
p53/ fibroblast during normal cell division. The cell
rounded up from the coverslip (Fig. 2A, 5:21 PM), underwent cytokinesis
(Fig. 2A, 5:32 PM), and completed division into two daughter cells
(Fig. 2A, 5:40 PM). Thus, the length of mitosis, as determined visually by rounding of the parent cell and separation into two daughter cells,
was approximately 20 min. Normal mitosis lasted 26 ± 8 (mean ± standard deviation) min in wild-type MEFs and 27 ± 7 min in
p53/ MEFs, based on observations made for at least 15 cells of each genotype (data not shown). For our next experiments, we
used time-lapse techniques to determine the fate of fibroblasts that
initiated mitosis while in the presence of the spindle inhibitor
nocodazole. Figure 2B shows two nocodazole-treated p53+/+
fibroblasts that entered mitosis within the same 30-min window. Each
cell rounded up from the coverslip as was observed for normal mitosis
(Fig. 2B, 1:14 PM and 1:43 PM). However, the cells then changed shape
rapidly in apparent attempts to complete cell division over a period of
approximately 4 h (Fig. 2B, 3:33 PM and 4:31 PM). Strikingly,
after this time, the cells flattened again without completing mitosis
(Fig. 2B, 5:33 PM and 6:02 PM). This behavior of exiting from mitotic
arrest into an apparent interphase state without actually completing
mitosis, which we will refer to as adaptation, has been previously
described (24, 35). We also examined the behavior of
p53/ fibroblasts that entered mitosis while in the
presence of nocodazole. A representative p53/
fibroblast that initiated mitosis while growing in nocodazole is shown
in Fig. 2C. Like the p53+/+ cells, the p53/
fibroblast rounded up (Fig. 2C, 10:08 AM), changed morphology over a
4-h period (Fig. 2C, 11:00 AM and 12:17 PM), and then flattened back
out again onto the coverslip (Fig. 2C, 1:47 PM and 2:13 PM).
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Figure 2D shows quantitation of the time spent at mitotic arrest for
individual p53+/+ and p53/ MEFs that were
followed by time-lapse videomicroscopy. The length of arrest was
determined for at least 60 cells of each genotype. Both
p53+/+ and p53/ MEFs spent a much longer
period of time in a rounded mitotic state when they were grown in
nocodazole-containing medium than when they were grown in normal
medium. The average time spent arrested at mitosis under these
conditions was 4.4 h for p53+/+ MEFs and 4.6 h
for p53/ MEFs compared to 26 to 27 min normally spent
at mitosis in untreated cells. Cells of each genotype subsequently
exited from mitosis and flattened onto the coverslip without completing
cytokinesis. Interestingly, several cells of each genotype were
observed to undergo prolonged mitotic arrest of 8 to 14 h prior to
adaptation (Fig. 2D). These data demonstrate that p53 does not act as a
mitotic checkpoint per se, because p53+/+ and
p53/ MEFs behave identically at mitosis in the presence
of nocodazole.
Given that p53 does not affect mitotic arrest kinetics, we wished to
identify the stage at which p53 is required for cell cycle arrest
following nocodazole treatment. To address this question, individual
p53/ MEFs treated with nocodazole were followed through
mitotic entry and arrest, mitotic exit/ adaptation, and S phase entry.
Cells were plated onto gridded coverslips and followed by time-lapse videomicroscopy as described above. After 18 to 22 h of treatment, cells were pulsed with BrdU for an additional 4 h in the presence of nocodazole and then analyzed by immunofluorescence. Figure 3A shows a representative
p53/ MEF that attempted mitosis, adapted, and then
incorporated BrdU. The cell entered mitosis at 12 AM, remained in
mitotic arrest for several hours, and then exited mitosis at 5:30 AM.
BrdU label was added at 11 AM, and the cell was fixed 4 h later.
Immunofluorescent detection for BrdU uptake revealed that the cell had
been in S phase during the time the BrdU label was present, which was
5.5 to 9.5 hr after adaptation had occurred. Ten individual
p53/ fibroblasts were tracked with this method to
determine the number of hours which elapsed between morphological
adaptation and S phase entry (Fig. 3B). Importantly, cells did not
enter S phase immediately after exiting mitosis, as cells labeled with
BrdU 0 to 3.5 h postadaptation failed to stain positively. The
p53/ MEFs began to incorporate BrdU in a time window
approximately 4 to 6 h postadaptation. One p53/
MEF incubated with BrdU for 9 to 13 h after it had undergone adaptation failed to incorporate label. The behavior of this fibroblast does not necessarily indicate the time limit for the reentry of p53/ MEFs into S phase, however, because the fibroblast
may either already have completed S phase at the time of the BrdU label
or may not yet have initiated it. These data show that p53 is required to prevent entry into S phase beginning in a specific time interval after nocodazole-treated MEFs have attempted mitosis and subsequently adapted into an interphase state.
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p53 has been shown to prevent cells in G1 from entering S
phase following DNA damage (20, 21, 23). This arrest is
mediated in large part by p21, a transcriptional target of the p53
protein which inhibits CDKs and prevents phosphorylation of the Rb
protein, a requirement for S phase entry (2, 7). To test
whether a similar mechanism might be functioning to prevent S phase
entry in nocodazole-treated adapted cells, we performed immunoblot
analysis for p21 protein on wild-type and p53/ MEFs
treated with nocodazole (Fig. 4A). In
wild-type cells, levels of p21 protein increased after 16 h of
nocodazole treatment and remained elevated over a 48-h period. No p21
protein was detectable in nocodazole-treated p53/ MEFs.
These observations suggested a possible role for p21 in maintaining
arrest following nocodazole treatment. To establish whether p21 was
required for the arrest following spindle disruption, wild-type,
p53/, and p21/ MEFs were treated with
nocodazole for 24 h and analyzed by flow cytometry (Fig. 4B). As
was observed in Figure 1, wild-type MEFs arrested with 4N DNA content,
while p53/ MEFs continued to increase in ploidy.
p21/ MEFs were observed to have a phenotype similar to
that of p53/ MEFs, with a significant fraction of cells
undergoing an additional round of S phase to become 8N in DNA content.
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To demonstrate further that p21/ cells were capable of
reentry into S phase following nocodazole treatment,
p21/ MEFs were treated with nocodazole for 24 h,
pulsed with BrdU while still in the presence of nocodazole for another
4 h, and analyzed by immunofluorescence (Fig. 4C). A high
percentage of nocodazole-treated p21/ MEFs were found
to incorporate BrdU under these conditions, indicating inappropriate S
phase entry. Similar results were observed when wild-type,
p53/, and p21/ MEFs were treated with
the microtubule-destabilizing drug colcemid, demonstrating the
generality of this response (data not shown). Quantitation of the
percentage of wild-type, p53/, and p21/
MEFs that stained positively for BrdU incorporation after 28 h of
nocodazole treatment is presented in Fig. 4D. Approximately 54% of
p21/ MEFs incorporated BrdU during the labeling period
compared to 65% of p53/ MEFs and 14% of wild-type
MEFs. Untreated MEFs of all three genotypes had similar percentages of
cells in S phase (approximately 50 to 60%; data not shown). Thus, the
p21/ MEFs had an abnormal arrest phenotype which was
similar to that of p53/ MEFs, although not as severe.
Given these results, we concluded that p21 is required to fully prevent
cells treated with spindle drugs from reentering S phase.
Thus far, our data demonstrated that p53 functions to prevent S phase entry following mitotic arrest and adaptation. However, while inappropriate S phase entry had been observed by FACS analysis to occur in thousands of cells, mitotic arrest and adaptation had been observed in only approximately 100 cells in these experiments. To demonstrate that mitotic arrest and adaptation also occurred on the level of entire populations of cells, we turned to methods other than time-lapse videomicroscopy. NIH 3T3 cells were used in these experiments for ease of synchronization and to show that our previous observations did not reflect a phenotype specific to our MEF clones. The NIH 3T3 cell line has been shown to contain wild-type p53 (16, 29, 31, 36, 40). NIH 3T3 cells were synchronized for 48 h in low serum medium and then released into high serum medium with or without nocodazole. At 6-h time points, cells were photographed and then fixed for flow cytometric analysis of DNA content (Fig. 5). At the time of release into high serum, cells were synchronized with a 2N DNA content (Fig. 5A, untreated, and B, nocodazole-treated; 0 h). Cells entered S phase synchronously (Fig. 5A and B, 14 h) and completed DNA synthesis by 20 h after release into high-serum-containing medium (Fig. 5A and B, 20 h). The presence of nocodazole did not noticeably delay progression into or completion of S phase, as judged by DNA content. The first mitoses were visible at the same time point in photographs of untreated and nocodazole-treated cells as rounded refractile cells (Fig. 5A and B, 20 h). Within 6 h, all of the cells in the untreated population had completed mitosis, as the 4N population had been replaced by cells with 2N and S phase DNA content (Fig. 5A, 26 h). In contrast, all of the cells in the nocodazole-treated population had a 4N DNA content at 26 h, and most appeared to be rounded up and arrested at mitosis (Fig. 5B, 26 h). By the next time point, however, these cells had flattened out again but still had a 4N DNA content, indicating their failure to complete mitosis (Fig. 5B, 32 h). In the adapted cells, microtubules were depolymerized and chromatin was decondensed, as determined by indirect immunofluorescence for tubulin and nuclear staining with DAPI, respectively (data not shown). Thus, as observed with wild-type MEFs, NIH 3T3 cells did not undergo an additional round of DNA synthesis during nocodazole treatment. These results indicate that the great majority of murine fibroblasts in a synchronized population undergo mitotic arrest and adaptation when treated with nocodazole.
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We observed some similarities between the p53-dependent checkpoint following nocodazole treatment and the p53-dependent G1 checkpoint following irradiation. Our data showed that a primary effector of the G1 checkpoint response, p21, was also required for arrest after spindle disruption. Also, adapted cells had the flattened appearance and decondensed chromatin characteristic of cells in G1. To determine whether adapted cells were similar to G1 cells at the molecular level, we measured expression of cyclin B1, a mitotic cyclin, and cyclin E, a G1 cyclin, in nocodazole-treated cells. Cyclin B1 is expressed at high levels in mitotic cells and declines when cells enter G1, while cyclin E expression is highest in cells in the late G1 phase of the cell cycle (9, 26). NIH 3T3 cells were synchronized in low serum, released into medium with high serum plus nocodazole, and collected at time points for immunoblotting. The blot was probed with antibodies to cyclin B1 and cyclin E (Fig. 6). Cyclin B1 levels peaked at 24 h and decreased afterward. The appearance of this peak corresponded with the presence of mitotic cells (data not shown). Cyclin E levels were elevated at 16 h as cells progressed from G1 into S phase and then decreased with the onset of mitosis. At 32 h, mitotic-arrested cells had just adapted into a flattened state (data not shown) and cyclin E levels remained low (Fig. 6). Cyclin E levels then increased again in the adapted cells at 40 h (Fig. 6). Thus, adapted cells express high levels of cyclin E, a G1 marker, despite having a DNA content characteristic of cells in the G2 or M phase of the cell cycle. The delayed expression of cyclin E relative to adaptation may reflect the fact that cyclin E is normally expressed late in G1 and that adapted cells take several hours to progress to this state.
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To characterize further the nocodazole-induced adapted state, we examined adapted cells for another marker of G1, hypophosphorylated Rb protein. In the G1 phase of the cell cycle, the Rb protein (pRB) is in a hypophosphorylated state (3, 4, 6, 32). As part of the G1-to-S-phase transition, pRB becomes hyperphosphorylated on multiple sites and remains so until anaphase of mitosis (30). We analyzed serum-synchronized, nocodazole-treated NIH 3T3 cells at various time points for their pRB phosphorylation status (Fig. 7A) and DNA content (Fig. 7B). At time 0, cells had been serum starved for 48 h and were 2N in DNA content. All pRB was hypophosphorylated, as would be expected for cells in G0. Sixteen hours after release into medium containing high serum plus nocodazole, pRB had become hyperphosphorylated, corresponding with the entry of cells into S phase. At 24 h postrelease, cells had completed DNA synthesis and were entering mitotic arrest, from which they later adapted (Fig. 7B and data not shown). By 40 h of treatment, cells had adapted and pRB was predominantly in the hypophosphorylated state. Therefore, like cyclin E expression, pRB phosphorylation status in adapted cells resembles the pattern seen in G1 cells. Because pRB is present in adapted cells in the active, or hypophosphorylated, state, it is possible that like p53 and p21, pRB functions to prevent S phase reentry during nocodazole treatment. Such a mechanism seems likely, given that p21 is required for arrest in nocodazole-treated cells and that p21 induces cell cycle arrest in part by inhibiting pRb phosphorylation.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Many functions have been attributed to the p53 protein, which is
clearly a critical cell cycle regulator in mammalian cells (25). In this study, we have addressed the question of
whether the ability of cells to initiate and maintain the mitotic
spindle checkpoint is affected by their p53 status. We have attempted to clarify the role of p53 at mitosis by monitoring the response of
individual p53+/+ and p53/ MEFs to spindle
inhibitors. In order for p53 to qualify as a true mitotic checkpoint,
p53+/+ and p53/ MEFs would have to behave
differently at mitosis. However, we observed that both wild-type and
p53-deficient MEFs arrested at mitosis following disruption of the
spindle and that cells of either genotype arrested for the same length
of time and behaved identically during the mitotic arrest. This
behavior is opposite to that of mammalian cells in which the MAD2 or
BUB1 mitotic checkpoint gene products, which monitor the integrity of
the mitotic spindle, have been inhibited. Recent studies have
identified a human homolog for the S. cerevisiae MAD2
checkpoint gene, hsMAD2, as well as a murine homolog of the S. cerevisiae BUB1 checkpoint gene. As would be predicted from
experiments with S. cerevisiae mad strains, mammalian cells
electroporated with an anti-hsMAD2 antibody did not undergo arrest at
mitosis when the cells were treated with nocodazole (28).
Similarly, when treated with nocodazole, mammalian cells which
expressed a dominant negative version of the murine Bub1 gene arrested
at mitosis much less frequently than their normal counterparts
(39). Thus, when contrasted with genes which are known to
act at mitotic checkpoints in mammalian cells, p53 acts in a different
manner. We can conclude that, unlike the hsMAD2 and murine Bub1 genes,
the presence or absence of p53 has no impact on the ability of cells to
detect microtubule disruption and initiate metaphase arrest. Thus, we
have shown definitively that p53 does not act to implement a checkpoint
at mitosis in mouse embryonic fibroblasts.
We observed that p53 does have a checkpoint function in MEFs following
nocodazole treatment, specifically, to prevent S phase reentry
following adaptation from mitotic arrest. Consistent with this finding
is the observation that levels of p53 protein and its downstream target
gene product p21 both increase following treatment with nocodazole or
other microtubule drugs (Fig. 4A) (33, 40). Importantly, we
have found that the downstream arrest mechanism activated by p53
requires the CDK inhibitor p21, as p21/ MEFs fail to
arrest in response to nocodazole treatment and enter another round of S
phase, in contrast to the results of an earlier study (7).
Although our data were obtained with cells treated with nocodazole, we
obtained similar results with the spindle inhibitor colcemid (data not
shown), which was the microtubule-destabilizing drug used in the
earlier study. We eliminated the possibility that the p21 protein
itself was acting at a mitotic checkpoint, because like wild-type and
p53/ MEFs, p21/ MEFs treated with
nocodazole arrested at mitosis for 4 to 5 h and then adapted (data
not shown). The simplest explanation for our observations is that in
adapted cells, activation of p53 leads to increases in the level of p21
protein, which then initiates cell cycle arrest by inhibiting
cyclin-CDK activity. Interestingly, the contribution of p21 to the
arrest response following nocodazole treatment and adaptation is
proportionately similar to the contribution of p21 to the
G1 arrest response following DNA damage. In
nocodazole-treated, adapted MEFs, p21 is responsible for about 80% of
the p53-mediated arrest response, while in irradiated MEFs, p21
accounts for approximately 70 to 80% of the p53-dependent arrest in
G1 (2, 7). These data demonstrate not only that
p21 is required for the arrest following nocodazole treatment but also
that the requirement for p21 is quantitatively similar to the
requirement for p21 in the G1 arrest following DNA damage.
Taken together, our results suggest that p53 could be acting as a component of a G1 checkpoint in nocodazole-treated cells. First, p53 function is required to prevent reentry into S phase following checkpoint activation, as occurs in the G1 checkpoint following DNA damage. Second, p21 is required to execute this arrest and to an extent similar to its role in the G1 arrest checkpoint. Finally, cells which have exited mitotic arrest have the flattened appearance and decondensed chromatin of G1 cells and express the G1-specific markers cyclin E and hypophosphorylated pRB. Thus, p53 could be implementing a G1-like arrest in 4N adapted cells.
An interesting and as-yet-unanswered question is the nature of the upstream signal which triggers p53-dependent arrest following nocodazole treatment. One possibility is that as during the normal G1 checkpoint, p53 detects DNA damage. It is readily conceivable that a cell which attempted to undergo chromosome separation at metaphase when its spindle was destabilized by nocodazole could incur DNA damage, which would then be detected following exit from mitotic arrest. In this case, both the upstream and downstream signals for arrest following nocodazole treatment would be identical to those for G1 arrest following irradiation. Alternatively, p53 could be activated by other types of signals, such as the presence of two centrosomes in a G1-like cell. Another possible signal could be the presence of excess chromosomes in a G1-like cell, which would trigger a p53-dependent checkpoint to prevent endoreduplication. Our data do exclude one possibility, however, which is that p53 is activated in response to a loss of microtubule stability induced by nocodazole treatment. Our data on NIH 3T3 cells, which contain functional p53, show that like other cell types, they progress unhindered from G1 to mitosis while in the constant presence of nocodazole (Fig. 5) (19). Were p53 detecting a loss of microtubule stability in G1 or G1-like cells, these cells would never have progressed through S phase but instead would have arrested shortly after entry into G1.
While it has been speculated to act at multiple different cell cycle checkpoints, the p53 protein appears to have overlapping checkpoint functions following irradiation and spindle disruption. After irradiation, cells undergo p53-dependent arrest in G1 (20, 21, 23); after nocodazole-induced disruption of the mitotic spindle, cells arrest transiently at mitosis, adapt into a G1-like state, and then undergo p53-dependent arrest (33). In both instances, the p53-mediated arrest requires the CDK inhibitor p21 (2, 7). Thus, both the stage of the cell cycle at which p53 induces arrest and the downstream mechanism by which it implements arrest are similar after either irradiation or nocodazole treatment. In cells which lack functional p53 protein, failure to arrest following irradiation or nocodazole treatment would be predicted to lead to two distinctly different outcomes of chromosomal damage and polyploidy, respectively. Thus, the loss of p53 checkpoint function during tumorigenesis could lead to decreased genomic stability by multiple mechanisms.
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ACKNOWLEDGMENTS |
|---|
We thank J. Brugarolas for p21/ MEFs and L. Attardi for critical reading of the manuscript.
J.S.L. was supported in part by a predoctoral fellowship from the Office of Naval Research. T.J. is an Associate Investigator of the Howard Hughes Medical Institute.
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FOOTNOTES |
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* Corresponding author. Mailing address: MIT Center for Cancer Research, 40 Ames St., Building E17-517, Cambridge, MA 02139. Phone: (617) 253-0262. Fax: (617) 253-9863. E-mail: tjacks{at}mit.edu.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Mol Cell Biol, February 1998, p. 1055-1064, Vol. 18, No. 2
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