From the Consiglio Nazionale delle Ricerche Centre of
Evolutionary Genetics, Department of Genetics and Molecular Biology,
University of Rome "La Sapienza," Via degli Apuli 4, Rome 00185, Italy and the ** Molecular Oncogenesis Laboratory, Regina Elena Cancer
Institute, Rome 00158, Italy
Received for publication, October 18, 2000, and in revised form, February 20, 2001
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ABSTRACT |
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Growing evidence indicates a central role for p53
in mediating cell cycle arrest in response to mitotic spindle defects
so as to prevent rereplication in cells in which the mitotic division has failed. Here we report that a transient inhibition of spindle assembly induced by nocodazole, a tubulin-depolymerizing drug, triggers
a stable activation of p53, which can transduce a cell cycle inhibitory
signal even when the spindle-damaging agent is removed and the spindle
is allowed to reassemble. Cells transiently exposed to nocodazole
continue to express high levels of p53 and p21 in the cell cycle that
follows the transient exposure to nocodazole and become arrested in
G1, regardless of whether they carry a diploid or
polyploid genome after mitotic exit. We also show that p53 normally
associates with centrosomes in mitotic cells, whereas nocodazole
disrupts this association. Together these results suggest that the
induction of spindle damage, albeit transient, interferes with the
subcellular localization of p53 at specific mitotic locations, which in
turn dictates cell cycle arrest in the offspring of such defective mitoses.
Polyploidization is regarded as a crucial step leading to
aneuploidy in tumor cells (1, 2), which in turn is associated with high
malignancy and poor prognosis. A complex network of structural and
regulatory factors ensures that chromosomes segregate evenly in
daughter cells and prevents resumption of DNA synthesis before mitosis
has been completed. This process is subject to control
mechanisms collectively referred to as the post-mitotic checkpoint
(3).
A large body of data clearly documents the key role of the
p53 tumor suppressor gene in the G1/S checkpoint
in response to DNA damage, whereby DNA replication is prevented until
repair has occurred, or apoptosis is induced when repair is ineffective (reviewed in Ref. 4). Both p53 and its major regulatory target, the
cyclin-dependent kinase inhibitor p21, also act at later
cell cycle stages and are implicated in the G2 checkpoint,
which prevents mitotic entry following post-replicative DNA damage (5,
6). These p53-dependent regulatory pathways rest upon the
ability of p53 to act as a "sensor" of DNA damage and are now well
clarified. A more intricate aspect of cell cycle checkpoints is being
unraveled in a growing number of studies that indicate that p53 also
contributes to a checkpoint triggered in response to mitotic spindle
defects (3, 7, 8; reviewed in Ref. 9).
Nocodazole (Noc)1 is a well
known tubulin-depolymerizing agent that inhibits spindle assembly and
triggers a response known as the mitotic spindle checkpoint, following
which mitotic progression is arrested (10-12 and references therein).
In previous work (13) we sought to investigate the requirement of p53
activity in response to failure of the mitotic spindle. Two
human isogenic cell lines, i.e. the K562 (p53-negative)
erythroleukemia cell line and a parvovirus-resistant derivative clone
named KS, which reexpresses p53, were continuously exposed to Noc.
Human lymphoblastoid AHH1 cells were also examined in parallel
experiments. We found that the activation of the proper spindle
checkpoint, revealed by a delay in mitotic progression in the presence
of spindle damage, was independent of p53. However, mitotic arrest was
transient, and, after a delay of variable length, cells achieved
mitotic exit even in the absence of spindle (mitotic slippage). At that
stage, the cell cycle patterns of cell lines differed dramatically
depending on their p53 status; only p53-deficient K562 cells progressed
through the cell cycle in the presence of Noc, resuming DNA synthesis
and hence entering a polyploid cell cycle. On the contrary,
p53-expressing KS and AHH1 cells stopped cell cycle progression after
the first mitotic round, degraded cyclin B, and arrested in the
following G1 phase with a 4C DNA content. These results
converge with data obtained by other groups (reviewed in Ref. 9)
indicating that G1 arrest in tetraploid cells (post-mitotic
checkpoint) is p53-dependent.
In a further step to understand the mechanism of action of p53 in the
post-mitotic checkpoint, we examined the response of cells to transient
exposure to Noc, i.e. over the duration of one cell cycle
only. We report here that transient exposure to Noc generates a
prolonged signal for p53 activation, which persists during the first
cell cycle after Noc removal, i.e. when a functional spindle
is in fact allowed to reassemble. In addition, Noc alters the
subcellular localization of p53 during mitosis. Indeed, p53 associates
with centrosomes in normal mitotic cells; in contrast, this association
is disrupted by Noc and is not reestablished in cells that achieve
mitotic exit after transient exposure to Noc. Thus, the inhibition of
spindle assembly by Noc is reflected by the failure of p53 to associate
with structures of the mitotic apparatus; in turn, delocalization and
prolonged activation of p53 are associated with maintenance of a cell
cycle inhibitory signal in the G1 phase that follows the
removal of the spindle-damaging agent.
Cell Cultures--
Human cell lines AHH1 (non-transformed
lymphoblastoid cell line) and K562 (erythroleukemic cell line) were
grown in RPMI 1640 medium (Euroclone) supplemented with 10% fetal calf
serum in a 5% CO2 atmosphere at 37 °C. Exponentially
growing cultures were used for all experiments. Where indicated, AHH1
cells were transfected by electroporation (975 microfarads and
250 V) with a geneticin (G418)-resistant construct encoding a
temperature-sensitive version of the p53 protein (p53Val135), which at
38 °C acts as a dominant negative factor for wild-type p53. Control
cells in these experiments were generated by transfection with empty
vector carrying the G418 resistance gene alone, yielding the control
cell line henceforth designated AHH1neo.
Both transfected cell lines were kept under selection by culturing in
the presence of 500 µg/ml geneticin (Life Technologies, Inc.) and
maintained as polyclonal populations (AHH1dnp53
cells). Cell cultures were exposed to Noc for 20 h, then washed in
fresh medium, and cultured in Noc-free medium for the times indicated
in the text. Noc was dissolved in Me2SO (both from
Sigma) at a 1 mg/ml concentration. The required volume of 1 mg/ml Noc solution was directly added to exponentially growing cultures so as to
obtain a final concentration of 0.2 µg/ml.
Protein Extract Preparation and Western Immunoblotting
Assays--
Aliquots of 5 × 106 cells were withdrawn
from the cultures at the indicated times and centrifuged at low speed
at 4 °C. Pelleted cells were washed in ice-cold phosphate-buffered
saline containing 1 mM phenylmethylsulfonyl fluoride
(Sigma) and lysed as described (14). Cell lysates were centrifuged at
14,000 rpm at 4 °C for 30 min. Protein concentrations were
determined using the Bradford assay kit (Bio-Rad). Protein extracts
were resuspended in loading buffer (4% SDS, 100 mM
dithiothreitol, 0.5 mM EDTA, 20% glycerol, 100 mM Tris-HCl, pH 6.8, 0.1% bromphenol blue), boiled for 8 min, briefly centrifuged, and finally subjected to 10-12%
SDS-polyacrylamide gel electrophoresis. Electrophoresed proteins were
electrotransferred at 100 mA for 75 min onto a Trans-blot
nitrocellulose membrane (Schleicher & Schuell). Membranes were blocked
in 5% (w/v) low fat milk in TBST buffer (10 mM Tris, pH
8.0, 150 mM NaCl, 0.1% Tween 20) at 4 °C overnight and
then incubated for 1 h at room temperature with the following
primary antibodies in 5% milk, TBST: anti-cyclin B1 (GNS1, Santa Cruz
Biotechnology, sc-245), anti-p53 (DO-7, Dako, M7001), anti-p21 (C-19,
Santa Cruz Biotechnology, sc-397), anti-cyclin E (Ab-1, Calbiochem,
CC05). All primary antibodies were used at 0.5 µg/ml. Immunoreactive
proteins were detected using horseradish peroxidase-conjugated
secondary antibodies (Santa Cruz Biotechnology) and revealed using the
enhanced chemiluminescence system (ECL-plus, Amersham Pharmacia Biotech).
Flow Cytometry Analysis--
DNA replication was monitored
essentially as described (15) after addition of BrdUrd
(5-bromo-2'-deoxyuridine, 45 µM final concentration) to
the culture medium 30 min before harvesting the cells. Cells were fixed
in 70% ethanol (30 min, 4 °C), washed twice in 0.5% Tween 20 in
phosphate-buffered saline, and incubated in 3 M HCl for 45 min to denature the DNA. Cells were then exposed to anti-BrdUrd
monoclonal antibody (Dako clone Bu20a, M0744) and to secondary
fluorescein isothiocyanate (FITC)-conjugated antibody (Vector
Laboratories, FI2000) and were finally stained with propidium iodide.
Samples were analyzed using a FACStar (fluorescence-activated cell
sorter) Plus flow cytometer (Becton-Dickinson) and the WinMDI software. 10,000 events were recorded for each sample. The
amplification scale was logarithmic for FSC-H and FL1-H parameters and
linear for SSC-H, FL2-A, FL2-H, and FL2-W. Photomultiplier tension was set so as to place the peak corresponding to 2C DNA content
(G0/G1) at channel 200 in the FL2-H histogram.
A similar procedure, except for the HCl step, was adapted to measure
the DNA content and the fluorescence associated with anti-cyclin B1
(Santa Cruz Biotechnology), anti-p53 (see above for details), and MPM-2
(16; Dako M3514) antibodies. A negative control was also prepared for
each sample by treating the cells with a mouse nonspecific IgG and
secondary FITC-conjugated antibody (Santa Cruz Biotechnology). In all
types of analysis, cell aggregates were carefully gated out using FL2-W
versus FL2-A bivariant graphs.
Immunofluorescence Assays--
Cells were washed, spread on
glass coverslips by centrifugation, and fixed in 3.7% formaldehyde for
15 min at 4 °C. Cell preparations were permeabilized in 0.25%
Triton X-100 in phosphate-buffered saline for 5 min at room temperature
and 100% methanol for 10 min at p53-expressing Cells Undergo a Prolonged yet Reversible Arrest
after Transient Exposure to Noc--
In previous experiments designed
to analyze the mechanisms preventing polyploidization, we examined
human non-transformed AHH1 cells that were continuously cultured in the
presence of Noc. The cell cycle progressed normally until the first
mitotic division; after a transient mitotic delay, cells resumed
mitosis and eventually arrested in a G1-like tetraploid
state. This pattern was similar to that observed in the p53-proficient
KS cell line and differed from that of K562 cells, which lack
functional p53 and continue to progress into the cell cycle in the
presence of Noc (13).2
Thus, Noc activates a similar post-mitotic checkpoint pathway both in
AHH1 cells and in KS cells.
To further investigate the cell cycle inhibitory mechanisms triggered
by spindle failure, we examined cell cultures that were exposed to Noc
for a length of time allowing one cell division and subsequently
cultured in the absence of Noc. AHH1 and K562 cells were exposed to Noc
for 20 h, then washed, and further incubated in Noc-free growth
medium. Cell cycle progression was examined by simultaneous FACS
analysis of BrdUrd incorporation into newly replicating DNA and of the
genomic content revealed by propidium iodide incorporation. In these
experiments both cell lines were effectively arrested in
G2/M in the presence of Noc. Upon Noc removal, both cell
lines rapidly exited mitosis; 2 h after the block release, about
50% of the cells had reached the G1 phase (Fig.
1A). Thereafter a dramatic
difference in cell cycle resumption was observed between cell lines;
20 h after Noc removal, DNA replication was actively resumed in
p53-deficient K562 cells, the cell cycle profile of which was similar
to that of asynchronously cycling cultures (Fig. 1A,
lower row). In contrast, AHH1 cultures remained blocked with
a similar cell cycle profile for at least 20 h after release from Noc arrest (Fig. 1A, upper row);
after 48 h some resumption of cell cycle progression began; only
72 h after Noc removal, roughly corresponding to the length of
three cell cycles in normal conditions, did AHH1 cells eventually
recover the typical distribution of asynchronously cycling cultures
(Fig. 1A). These data therefore depict a significant
difference between p53-proficient and p53-defective cells in the
ability to induce a durable arrest after spindle failure; this
difference is most evident 20 h after Noc release. By Western
immunoblotting assays (Fig. 1B), AHH1 cells accumulated p53
during Noc exposure as expected (13, 18); furthermore, p53 levels
peaked at 20 h after Noc removal and gradually declined thereafter
(Fig. 1B). The gene encoding p21 is the best characterized
transcriptional target of p53, and its expression is triggered by
several cell cycle-disrupting agents. Our experiments show that p21
protein levels also peak 20 h after release from Noc arrest and
substantially parallel the p53 pattern of expression. Thus, the
persistence of G1 arrest in AHH1 cultures in the first cell
cycle after removal of Noc coincides with the highest induction of the
p53/p21 pathway.
G1 Arrest after Transient Exposure to Noc Occurs in
Diploid and Tetraploid Cell Populations--
The expression of cell
cycle markers was examined to determine the molecular features
associated with the induction of prolonged arrest in AHH1 cell
cultures. In these experiments we focused on the interval of time
during which the checkpoint was effective, i.e. the first
cell cycle after Noc removal. A time course analysis of AHH1 cell
extracts by Western immunoblotting (Fig.
2A) shows that during the
first 20 h of culture in the presence of Noc, cells accumulate
cyclin B, as expected; subsequently, cyclin B becomes fully degraded
within the first 4 h of release and fails to be newly synthesized
for as long as 20 h after Noc release. Concomitantly, cells
gradually accumulate cyclin E during the first 4 h after Noc
removal, indicating that mitotic exit has occurred; cyclin E continues
to be highly expressed thereafter. Thus, only a minor fraction, if any,
of the AHH1 cells that had been seen to be arrested with a 4C DNA
content after 20 h from Noc removal (Fig. 1A,
upper row) are capable of reexpressing cyclin B, whereas
most of the cell population maintains high levels of cyclin E
expression (Fig. 2A). These results suggest that the 4C-containing subpopulation is in fact constituted by tetraploid cells
produced by mitotic slippage and arrested in a G1-like
state. To further confirm this interpretation, cyclin B expression was directly investigated in AHH1 cell populations separated for their DNA
content by biparametric FACS analysis (Fig. 2B). We also
investigated the expression of a subset of mitotic markers independent
of cyclins, i.e. the G2/M-specific
phosphoepitopes reactive to the MPM2 antibody (16). FACS analysis
unambiguously indicates that AHH1 cells exit mitosis after Noc removal,
as indicated by the disappearance both of cyclin B and of MPM2 antigens
in the entire cell population, regardless of the genome content. Thus,
exposure of AHH1 cells to Noc during one cell cycle induces a prolonged
arrest in the G1 phase of the following cell cycle, in cell
populations with a diploid or tetraploid content.
Functional p53 Is Required for Prolonged G1 Arrest in
Cells Released from Transient Exposure to Noc--
We and others
previously showed that the post-mitotic checkpoint activated in the
presence of Noc is p53-dependent (3, 5, 7, 8, 13). To
assess whether p53 activity was also implicated after release from Noc
arrest, we preliminarily used biparametric FACS analysis to determine
p53 immunoreactivity in AHH1 cell populations of different genome size;
we detected an increased p53 signal in the presence of Noc,
i.e. when the cell population is essentially
G2/M-arrested; after Noc removal p53 levels continued to
increase, consistent with the data in Fig. 1B, and 20 h after
removal most AHH1 cells in both the 2C and 4C subpopulations express
p53 (data not shown). These data strongly suggest that p53 is
functionally implicated in sustaining the prolonged arrest observed in
response to transient Noc exposure.
To directly assess that hypothesis, we constructed AHH1 cell lines
defective for p53 function. AHH1 cell cultures were stably transfected
with a neomycin-resistant expression vector directing the synthesis of
a dominant negative allele of p53 (dnp53) (19). The dnp53
protein acts in these cells (henceforth designated
AHH1dnp53) as a dominant negative mutant,
because it retains the ability to interact with the endogenous,
wild-type p53 protein and sequesters it into a functionally inactive
complex. AHH1dnp53 cells express significantly
higher steady-state levels of protein compared with both AHH1 and
AHH1neo cells, reflecting the exogenous
expression of mutant p53 from the transfected plasmid. FACS analysis
was employed to monitor cell cycle progression in AHH1 cell cultures
subjected to Noc block and release in the presence or absence of
functional p53. AHH1 cells transfected with vector alone (indicated as
AHH1neo cells) responded to transient Noc
exposure with a durable cell cycle arrest, revealed by the lack of
BrdUrd incorporation, that was indistinguishable from that of the
parental AHH1 cell line (Fig. 3,
upper row). On the contrary, the
AHH1dnp53 cell culture, in which the function of
endogenous p53 was impaired, failed to induce a post-mitotic arrest
(Fig. 3, lower row), similar to what we had previously
observed with the p53-defective K562 cell line (see Fig.
1A). Together, these data indicate that the G1
arrest that follows the transient inhibition of the mitotic spindle is
dependent on p53 function.
By Western immunoblotting (Fig.
4A, left panel)
both the parental (AHH1) and vector-transfected
(AHH1neo) cell cultures showed a progressive
increase in p53 levels during the first 20 h from Noc release. In
contrast, the AHH1dnp53 cell line failed to
up-regulate p53 levels after exposure to Noc.
In reverse transcriptase-polymerase chain reaction experiments p21
mRNA transcription was significantly up-regulated in AHH1 cells
during Noc block and release, but not in p53-deficient K562 cells under
similar conditions (data not shown). We directly examined the abundance
of the p21 protein in vector-transfected and mutant p53-transfected
AHH1 cells by Western blotting assays (Fig. 4A, right
panel). In AHH1 and AHH1neo cells, p21
levels increased in the presence of Noc and continued to further
increase during the first 20 h of release, substantially paralleling those of p53. In contrast, AHH1dnp53
cells failed to express comparably high levels of p21 when exposed to
Noc and after Noc removal. Actually, a low level of p21 induction was
observed at late release times (20 h after Noc removal), possibly reflecting the activity of residual wild-type p53 that had evidently not been completely inactivated by the dominant negative mutant. Microdensitometric analysis of the signal intensity (Fig.
4B) indicated, however, that p21 was induced only 2-fold and
3-fold after 8 and 20 h, respectively, of Noc release in
AHH1dnp53 cells, compared with 15- and 17-fold,
respectively, in AHH1neo cells. Together these
results indicate that p53-dependent G1 arrest
after spindle damage is associated with up-regulation of p21 protein levels.
Exposure to Noc Disrupts the Subcellular Localization of p53 in
Mitotic Cells--
The results thus far indicate that p53 is central
in mediating a prolonged cell cycle arrest that persists for several
hours after removal of the spindle-damaging agent, when spindle
assembly can actually be recovered. We therefore decided to investigate the intracellular distribution of p53 before and after induction of
spindle damage by indirect immunofluorescence.
In normal interphase cells, p53 shows a weak and diffuse pattern
throughout nuclei (Fig. 5A).
During mitosis, p53 is redistributed in a typical organization
consisting of discrete spots. Because the number and arrangement of p53
spots in mitotic cells resembled that of centrosomes, double
immunofluorescence experiments were performed using anti-p53 antibody
in combination with antibodies against centrosomal markers,
i.e. centrin, a major constituent of centrioles (20-21),
g-tubulin, forming the nucleating material (reviewed in Refs. 22-24),
and glutamylated tubulin, a specifically modified form of tubulin
associated with centriolar microtubules (17). Fig. 5, B and
C, shows results obtained with anti-centrin antibody in
normal mitotic cells; p53 spots are associated with or in close
proximity of centriole pairs visualized by centrin staining. Similar
results were obtained using two anti-p53 antibodies of different clonal
origin, in combination with antibodies against independent centrosomal
markers (see below).
We then examined cells exposed to Noc, i.e. typically
blocked in prometaphase and completing mitosis during the release from Noc arrest (Fig. 6). In Noc-exposed
prometaphase-arrested cultures the intensity of the p53 signal
increased compared with mitotic cells from asynchronous cultures; most
importantly, p53-positive spots failed to associate with centrosomes
(Fig. 6A) and were organized in variable numbers.
Furthermore, p53 was neither down-regulated in abundance nor
redistributed in its normal pattern after Noc removal, despite the
rapid resumption of mitotic progression; indeed, within 80 min from Noc
removal cells had progressed to ana-telophase (Fig. 6, B and
C), with a substantial recovery of their chromosome
segregation capability at each pole, and yet p53 spots continued to be
intense in fluorescence and variable in number and did not reassociate
with centrosomes. The abundance of p53 continued to increase even after
cells had completed mitosis and reached the following G1,
when p53 massively reentered the nucleus (Fig. 6D). To fully
assess the significance of these findings, we had to rule out the
possibility that the effect in Fig. 6 reflected merely an increase in
centriole splitting following Noc exposure. We first sought to quantify
the actual occurrence of centriole splitting in NOC-arrested
prometaphase cells by analyzing anti-centrin reactive spots. No
evidence of splitting was seen in 95 of 100 examined mitoses,
three cells showed more than two centriole pairs, and only in two cells
were centrioles abnormally separated; these figures are not
significantly different from mitotic figures from asynchronous
cultures. Immunofluorescence experiments were also repeated in cells
processed with anti-p53 and anti-g-tubulin antibodies (Fig.
7); these experiments confirmed that in
control mitotic cells p53 spots colocalize with g-tubulin, as indicated
by the yellow pseudocoloration of centrosomes (Fig. 7, A and
B). In contrast, in Noc-arrested and released cultures, the
association of p53 with centrosomes was again found to be inhibited
(Fig. 7, C and D). Similar patterns were observed
in cultures processed using an anti-glutamylated tubulin antibody (data
not shown).
Data are quantified in Table
I. In mitotic figures from Noc-exposed
and released cultures p53 spots were never found to be associated with
centrosomes. This contrasts with the typical pattern observed in
asynchronous control cultures, in which 80% of mitotic cells showed
centrosome-associated spots. In conclusion, therefore, these
results indicate that the stabilization and activation of p53 occurring
during exposure to Noc is accompanied by the lack of association of p53
with mitotic centrosomes, which persists during mitotic exit after Noc
removal.
It is now well established that mitotic spindle damage can
uncouple mitotic execution from DNA synthesis (rereplication), leading
cells to polyploidy and hence to an increased risk of neoplastic
transformation. Several works indicate that functional p53 is
implicated in regulatory networks that act to prevent polyploidization during cell exposure to spindle-damaging agents (3, 7, 8, 13, 25-26).
These studies reported a durable, if not irreversible, p53-dependent inhibition of rereplication. However, in all
tested experimental protocols, cells were continuously exposed to
mitotic spindle poisons (3, 8, 13). Under those conditions, p21 and pRb
can be viewed as downstream effectors of p53 acting to prevent
polyploidization in cells in which spindle assembly is continuously
inhibited (27-29).
In contrast, the role of p53 in the control of cell cycle progression
after removal of spindle-inhibiting drugs has never been examined
before. Here we have analyzed the response of cells transiently exposed
to Noc and have followed up the subsequent release in Noc-free medium.
The present results indicate that transient exposure to Noc triggers
the p53 pathway, which continues to operate in the following cell
cycle, at least in human non-tumorigenic hematopoietic cells, by
blocking the G1/S transition and preventing the following
round of DNA replication. Thus, mitotic impairment by spindle
inhibition generates a prolonged signal that is maintained throughout
cell division in the following cell cycle. The prolonged G1
arrest implicates p21 up-regulation at the gene and protein levels, and hence, presumably, cdk inhibition.
By combining cytometry and immunofluorescence analysis, we have found
that the p53 signal is first switched on in prometaphase-arrested cells
by Noc, i.e. before chromosome segregation is completed, and
hence before any genomic imbalance due to missegregation can be
detected in daughter cells. Cell cycle arrest remains effective for at
least 20 h after Noc release in AHH1 but fails in the p53 Noc acts with a well established mechanism of action as a
non-clastogenic, purely microtubule-directed drug (31; see Ref. 32 for
a review). In conditions similar to those used in the present
study, Noc lacks any DNA-damaging activity in comet
assays.3 Thus, there
are grounds to rule out the possibility that p53 induction by Noc
reflects a DNA-damaging side effect of the drug rather than genuine
spindle damage. In addition, the recent observation that p53
stabilization in response to Noc is mediated by phosphorylation events
that are distinct from those induced by DNA damage (18) further
suggests that the pathway(s) of p53 induction in response to Noc are
specific and depend on transduction cascades distinct from those
responsive to DNA damage.
Because Noc primarily or exclusively targets tubulin (33, 34), and yet
spindle assembly is rapidly resumed upon Noc removal, giving rise to
segregation of chromosomes to the poles (see Figs. 6 and 7), two major
scenarios may be envisaged for the activation and maintenance of the
p53-dependent checkpoint after transient exposure to Noc.
The checkpoint might be evoked in response to the possible occurrence
of aneuploid cells during mitotic exit after Noc removal, or the
failure of the spindle structure might directly be sensed as a trigger
for p53 activation. Although these scenarios are not necessarily
mutually exclusive, in our opinion several considerations favor the
latter possibility. First, in our experiments both the diploid and
tetraploid cell populations, the latter being generated during mitotic
slippage in the presence of Noc, progress through the
mitosis-to-G1 transition within 2 h from Noc removal,
as judged from the disappearance of mitotic markers (cyclin B- and
MPM-reactive antigens) from the entire cell population and the
resumption of G1 cyclin synthesis (Fig. 2). This molecular
pattern is stabilized during the first 20 h after Noc removal
(Fig. 2). At this time, the p53 signal reaches its highest level in the
cell population (Fig. 1), with a comparable intensity in the diploid
and tetraploid cell populations by FACS analysis. These results
indicate that G1 arrest takes place both in cells that
achieve chromosome segregation and in cells that undergo mitotic
slippage. This would suggest that the genome size does not per
se constitute the signal for p53 activation. Furthermore, the
trigger for p53 stabilization (Figs. 6 and 7) and transcriptional activation of the p21-encoding gene (Fig. 4) is already effective during Noc exposure, i.e. before chromosome segregation
occurs and before any genomic imbalance can be detected in daughter
cells. It remains possible that the occurrence of aneuploid cells
contributes to sustain the p53 signal; as a whole, however, the present
observations are consistent with the view that mitotic spindle failure,
rather than chromosome missegregation, triggers
p53-dependent arrest in the cell cycle that follows spindle damage.
Spindle-targeting drugs including taxol, vincristin, and Noc have
recently been shown to provoke p53 stabilization by inducing specific
phosphorylation events (18), implying that the spindle microtubules
activate direct and specific pathways culminating with the induction of
p53 activity. Consistent with this view, a recent study has highlighted
p53 features that may disclose novel p53-dependent
signaling mechanism(s). (i) p53 associates with microtubules in
vitro and in vivo; (ii) this association is mediated by
a specific domain responsible for the association of p53 with a and b
tubulin and requires microtubule integrity and dynamics; (iii) p53 is
transported along microtubules in a dynein-dependent manner
(35). Although these observations are essentially derived from studies
of interphasic tubulin and microtubules, their implications can be of
great relevance to the understanding of the role of p53 in mitosis.
Microtubule polymerization inhibitors interfere with the subcellular
localization of activities such as NuMA, which after nuclear
envelope breakdown relocates from nuclei to the spindle poles via
dynein-dependent transport along microtubules (36).
Similarly, inhibition of spindle assembly by Noc during mitosis may
interfere with the localization of p53 to specific structures of the
mitotic apparatus. In this work, we found that p53 normally localizes
with or in the immediate proximity of the spindle pole component
represented by centrosomes in mitotic cells (Fig. 5). This association
is disrupted in the presence of Noc and is not reestablished in cells
that complete mitosis after Noc removal (Figs. 6 and 7).
An increasing body of evidence now indicates that centrosomes act as
pivotal structures in cell cycle regulatory networks; they actively
participate in the cell division regulatory machinery (37 and
references therein), and their own biogenesis and duplication are
controlled by major cell cycle regulators, including
cyclin-dependent kinases (38-42) and p53 itself (43-46).
Indeed, impairment of p53 function results in centrosome
overduplication (46, 47). Furthermore, in cells transformed with the
adenovirus early gene product E1B, p53 is sequestered to cytoplasm, and
its association with centrosomes during mitosis is prevented (43),
indicating that p53 dysfunction can target the organization or activity
of centrosomes at least in certain transformed cell types.
Subcellular localization is crucial to p53 function (35, 43, 48; for
reviews see also Refs. 9, 49, and 50). It is tempting to hypothesize
that the timely association of p53 with centrosomes constitutes an
important regulatory step during the mitotic division. The present
results indicate that p53 function, initially triggered by Noc, does
not operate to arrest mitotic progression itself but becomes effective
during the following cell cycle. Because p53 continues to be
up-regulated in G1, i.e. after the recovery of the spindle, we would like to suggest that the absence, albeit transient, of a functional spindle generates the signal that triggers the "mitotic" activation of p53. The spindle is the essential structural component that allows mitotic completion. The inhibition, even temporary, of this structure can evidently interfere with signaling process(es) mediated by the spindle itself.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C. Fixed and permeabilized
cells were pre-incubated in 20% goat serum for 30 min at 37 °C in a
humidified chamber and then incubated for 2 h with anti-p53
antibody. Mouse monoclonal clone DO-7 (Dako M 7001, 1:100) was mostly
used (4 µg/ml); in certain experiments sheep polyclonal clone Ab-7
(Oncogene Science PC35, 1:200) was used. Antibodies to centrosomal
components included the following: rabbit anti-centrin antibody,
described in Ref. 17 (1:500 dilution), rabbit anti-
tubulin (Sigma
T3559, 1:2000 dilution), and anti-glutamylated tubulin (GT335), as
indicated in Ref. 17. Antibodies were diluted in 5% goat serum. After three washes in 0.05% Tween 20 in phosphate-buffered saline, cells were incubated with rhodamine-conjugated secondary antibody against centrin or
-tubulin antibody (Santa Cruz Biotechnology, sc2091, 1:400) for 30 min at 37 °C. After three more washes cells were incubated with FITC-conjugated secondary antibody (Vector
Laboratories FI2000, 1:300) directed against anti-p53 antibody
for 30 min. Cell spreads were counterstained with 0.2 µg/ml DAPI for
10 min at room temperature to stain the DNA and mounted on glass slides in Vectashield (Vector Laboratories). Cell preparations were
examined under an Olympus AX70 microscope equipped with
epifluorescence, and photographs were taken (× 100 objective) using a
cooled camera device (Photometrics).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Analysis of cell cycle progression after
transient exposure to Noc. A, biparametric FACS
analysis of BrdUrd incorporation (y axis) and of the DNA
content (x axis) in K562 (top) and AHH1
(bottom) cell lines exposed to Noc for 20 h, then
released in Noc-free medium, and followed up for 72 h. Panels are
representative of two independent experiments. B, Western
immunoblotting of p53 and p21 proteins in AHH1 cell cultures arrested
in Noc and released in Noc-free medium for the indicated times.
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Fig. 2.
AHH1 cells arrest in the G1 phase
of the cell cycle that follows transient exposure to Noc.
A, Western immunoblotting analysis of cyclins B and E in
AHH1 extracts from asynchronously cycling cultures (Asy),
from cells exposed to Noc for 20 h (Noc), or harvested
at the indicated times after Noc release. B, biparametric
FACS analysis of AHH1 cell populations separated for their DNA content
(x axis); levels of expression of cyclin B (top
panels) or MPM2-reactive antigens (bottom panels) are
plotted on the y axis. The base lines in the panels
correspond to the upper limit of nonspecific fluorescence. Percentages
of positive cells are indicated.
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Fig. 3.
Functional p53 is required for
induction of the post-mitotic checkpoint after transient exposure to
Noc. Upper row, stably transfected
AHH1neo (neomycin-resistant vector) cells;
lower row, stably transfected
AHH1dnp53 (neomycin-resistant construct
expressing the dominant negative Val-135 mutant of p53) cells; both
cell lines were exposed to Noc or harvested at the indicated times
after Noc release and then subjected to biparametric FACS analysis of
BrdUrd incorporation (y axis) and of the DNA content
(x axis). Panels are representative of three independent
experiments.
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Fig. 4.
Induction of the post-mitotic checkpoint in
response to transient exposure to Noc implicates p21
up-regulation. A, Western blot analysis of p53
(left panels) and p21 (right panels) in parental
(AHH1), vector-transfected (AHH1neo), and p53
dominant negative (AHH1dnp53) cell lines during
Noc arrest and release. B, quantification of the p21 signals
in immunoblots from AHH1neo (plain histograms)
and AHH1dnp53 (empty histograms) cell extracts.
After exposure, autoradiographs of ECL-processed p21 immunoblots were
scanned by microdensitometry. Values obtained in each time point after
Noc exposure and release were normalized relative to the p21 signal
measured in asynchronously cycling cells and hence indicate the extent
of p21 induction by Noc (represented by histograms). Asy,
asynchronous.
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Fig. 5.
Subcellular localization of p53 in
normal mitotic cells. A, interphase; B,
prometaphase; C, anaphase from asynchronously cycling AHH1
cultures after staining with DAPI to visualize chromosomes
(pseudocolored in blue, left column) and
processing for indirect immunofluorescence (middle column)
to localize centrin (Cen) (rhodamine-conjugated
secondary antibody, pseudocolored in red) and p53
(FITC-conjugated secondary antibody, pseudocolored in
green); superimposed signals appear in yellow.
Merged pictures are shown in the right column.
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Fig. 6.
Subcellular localization of p53 in
AHH1 cells after exposure to Noc. A, Noc-arrested
prometaphase cell; B, anaphase photographed 50 min after Noc
removal (the arrow indicates a lagging chromosome);
C, ana-telophase 80 min after Noc removal. DAPI-stained
chromosomes are pseudocolored in blue (left
column); centrin (Cen) and p53 were localized by
indirect immunofluorescence by rhodamine-conjugated
(pseudocolored in red) and FITC-conjugated (pseudocolored in
green) secondary antibodies, respectively (middle
column). Merged pictures are shown in the right
column.
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Fig. 7.
Colocalization of p53 and
centrosomes is lost in Noc-exposed cells. A, metaphase;
B, anaphase from asynchronously cycling AHH1 cell cultures;
C, Noc-arrested prometaphase cell; D, anaphase
exiting mitosis 90 min after Noc release. Chromosomes are visualized by
DAPI staining (pseudocolored in blue, left
column). tubulin and p53 are pseudocolored in red
(rhodamine-conjugated secondary antibody) and green
(FITC- conjugated secondary antibody), respectively, in the
middle column. Merged pictures are shown in the right
column. The yellow signal in A and
B indicates that p53 colocalizes with centrosomes in normal
mitotic cells but not in Noc-exposed or released cells.
Association of p53 with centrosomes in AHH1 cell cultures during
mitosis
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
K562 cell line (Fig. 1A) and in AHH1 cell populations
expressing a dominant negative mutant of p53 (Fig. 3), indicating that
prolonged cell cycle arrest is clearly p53-dependent. At
later times, i.e. 72 h post-release, arrest-proficient
AHH1 cells eventually resume cell cycle progression (Fig.
1A); thus, a typical checkpoint (30) is reversibly activated
in response to mitotic spindle failure. The prolonged p53 signal
and ensuing cell cycle arrest detected in the present study in response
to transient spindle inhibition represent novel aspects of the
post-mitotic checkpoint and raise two major questions: (i) the nature
of the cell cycle arrest-triggering signal and (ii) the pathway through
which the arrest signal is maintained in daughter cells after mitotic exit.
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ACKNOWLEDGEMENTS |
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We thank Antonella Palena and Alessandra Tritarelli for their contributions to this work and Silvia Bonaccorsi, Barbara Di Fiore, and Franco Tatò for helpful comments and advice on this manuscript. We are also grateful to Micheline Kirsch-Volders and Berlinda Verdoodt (University of Bruxelles, Belgium) for communicating unpublished results.
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FOOTNOTES |
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* This work was supported by Consiglio Nazionale delle Ricerche.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
¶ Supported by a fellowship from the Italian National Institute of Health (Istituto Superiore di Sanità).
Present address: Dept. of Cell Biology, Max Planck Institute
of Biochemistry, Am Klopferspitz 18a, D-82152 Martinsried, Germany.
To whom correspondence should be addressed. Tel.: 39 06 4457528; Fax: 39 06 4457529; E-mail: ecundari@eudoramail.com.
Published, JBC Papers in Press, February 28, 2001, DOI 10.1074/jbc.M009528200
2 M. Ciciarello, R. Mangiacasale, and E. Cundari, unpublished data.
3 B. Verdoodt and M. Kirsch-Volders, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: Noc, nocodazole; BrdUrd, 5-bromo-2'-deoxyuridine; FITC, fluorescein isothiocyanate; FACS, fluorescence-activated cell sorter; DAPI, 4',6-diamidino-2-phenylindole.
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---|
1. | Pihan, G. A., and Doxsey, S. J. (1999) Semin. Cancer Biol. 9, 289-302[CrossRef][Medline] [Order article via Infotrieve] |
2. | Vessey, C. J., Norbury, C. J., and Hickson, I. D. (1999) Prog. Nucleic Acid Res. Mol. Biol. 63, 189-221[Medline] [Order article via Infotrieve] |
3. |
Lanni, J. S.,
and Jacks, T.
(1998)
Mol. Cell. Biol.
18,
1055-1064 |
4. | Bates, S., and Vousden, K. H. (1996) Curr. Opin. Genet. Dev. 6, 12-18[Medline] [Order article via Infotrieve] |
5. |
Bunz, F.,
Dutriaux, A.,
Lengauer, C.,
Waldman, T.,
Zhou, S.,
Brown, J. P.,
Sedivy, J. M.,
Kinzler, K. W.,
and Vogelstein, B.
(1998)
Science
282,
1497-1501 |
6. | O'Connell, M. J., Walworth, N. C., and Carr, A. M. (2000) Trends Cell Biol. 10, 296-303[CrossRef][Medline] [Order article via Infotrieve] |
7. | Cross, S. M., Sanchez, C. A., Morgan, C. A., Schimke, M. K., Ramel, S., Idzerda, R. L., Raskind, W. H., and Reid, B. J. (1995) Science 267, 1353-1356[Medline] [Order article via Infotrieve] |
8. | Minn, A. J., Boise, L. H., and Thompson, C. B. (1996) Genes Dev. 10, 2621-2631[Abstract] |
9. | Meek, D. W. (2000) Pathol. Biol. 48, 246-254[Medline] [Order article via Infotrieve] |
10. | Rieder, C. L., and Salmon, E. D. (1998) Trends Cell Biol. 8, 310-318[CrossRef][Medline] [Order article via Infotrieve] |
11. | Kirsch-Volders, M., Cundari, E., and Verdoodt, B. (1998) Mutagenesis 13, 321-335[Abstract] |
12. |
Verdoodt, B.,
Decordier, I.,
Geleyns, K.,
Cunha, M.,
Cundari, E.,
and Kirsch-Volders, M.
(1999)
Mutagenesis
14,
513-520 |
13. | Casenghi, M., Mangiacasale, R., Tuynder, M., Caillet-Fauquet, P., Elhajouji, A., Lavia, P., Mousset, S., Kirsch-Volders, M., and Cundari, E. (1999) Exp. Cell Res. 250, 339-350[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Di Fiore, B.,
Guarguaglini, G.,
Palena, A.,
Kerkhoven, R. M.,
Bernards, R.,
and Lavia, P.
(1999)
J. Biol. Chem.
274,
10339-10348 |
15. |
Battistoni, A.,
Guarguaglini, G.,
Degrassi, F.,
Pittoggi, C.,
Palena, A.,
Di Matteo, G.,
Pisano, C.,
Cundari, E.,
and Lavia, P.
(1997)
J. Cell Sci.
110,
2345-2357 |
16. | Davis, F. M., Tsao, T. Y., Fowler, S. K., and Rao, P. N. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2926-2930[Abstract] |
17. |
Bobinnec, Y.,
Khodjakov, A.,
Mir, L. M.,
Rieder, C. L.,
Eddé, B.,
and Bornens, M.
(1998)
J. Cell Biol.
143,
1575-1589 |
18. | Stewart, Z. A., Tang, L. J., and Pietenpol, J. A. (2001) Oncogene 20, 113-124[CrossRef][Medline] [Order article via Infotrieve] |
19. | Michalovitz, D., Halevy, O., and Oren, M. (1990) Cell 62, 671-680[Medline] [Order article via Infotrieve] |
20. |
Paoletti, A.,
Moudjou, M.,
Paintrand, M.,
Salisbury, J. L.,
and Bornens, M.
(1996)
J. Cell Sci.
109,
3089-3102 |
21. |
Piel, M.,
Meyer, P.,
Khodjakov, A.,
Rieder, C. L.,
and Bornens, M.
(2000)
J. Cell Biol.
149,
317-330 |
22. |
Merdes, A.,
and Cleveland, D. W.
(1997)
J. Cell Biol.
138,
953-956 |
23. | Tassin, A. M., and Bornens, M. (1999) Biol. Cell 91, 343-354[CrossRef][Medline] [Order article via Infotrieve] |
24. | Schiebel, E. (2000) Curr. Opin. Cell Biol. 12, 113-118[CrossRef][Medline] [Order article via Infotrieve] |
25. | Wahl, A. F., Donaldson, K. L., Fairchild, C., Lee, F. Y. F., Foster, S. A., Demers, G. W., and Galloway, D. A. (1996) Nat. Med. 2, 72-79[Medline] [Order article via Infotrieve] |
26. |
Sablina, A.,
Iliynskaya, G. V.,
Rubtsova, S. N.,
Agapova, L. S.,
Chumakov, P. M.,
and Kopnin, B. P.
(1998)
J. Cell Sci.
111,
977-984 |
27. |
Pietenpol, J. A.,
Lengauer, C.,
Jordan, J.,
Kinzler, K. W.,
and Vogelstein, B.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
8390-8394 |
28. |
Dulic, V.,
Stein, G. H.,
Far, D. F.,
and Reed, S. I.
(1998)
Mol. Cell. Biol.
18,
546-557 |
29. |
Niculescu, A. B.,
Chen, X.,
Smeets, M.,
Hengst, L.,
Prives, C.,
and Reed, S. I.
(1998)
Mol. Cell. Biol.
18,
629-642 |
30. | Hartwell, L. H., and Weinert, T. A. (1989) Science 246, 629-634[Medline] [Order article via Infotrieve] |
31. | Jordan, M. A., Thrower, D., and Wilson, L. (1992) J. Cell Sci. 102, 401-416[Abstract] |
32. | Sorger, P. K., Dobles, M., Tournebize, R., and Hyman, A. A. (1997) Curr. Opin. Cell Biol. 9, 807-814[CrossRef][Medline] [Order article via Infotrieve] |
33. | Hoebeke, J., Van Nijen, G., and De Brabander, M. (1976) Biochem. Biophys. Res. Commun. 69, 319-324[Medline] [Order article via Infotrieve] |
34. |
Head, J.,
Lee, L. L. Y.,
Field, D. J.,
and Lee, J. C.
(1985)
J. Biol. Chem.
260,
11060-11066 |
35. | Giannakakou, P., Sackett, D. L., Ward, Y., Webster, K. R., Blagosklonny, M. V., and Fojo, T. (2000) Nat. Cell Biol. 2, 709-717[CrossRef][Medline] [Order article via Infotrieve] |
36. |
Merdes, A.,
Heald, R.,
Samejima, K.,
Earnshaw, W. C.,
and Cleveland, D. W.
(2000)
J. Cell Biol.
149,
851-862 |
37. | Zimmerman, W., Sparks, C. A., and Doxsey, S. J. (1999) Curr. Opin. Cell Biol. 11, 122-128[CrossRef][Medline] [Order article via Infotrieve] |
38. | Meraldi, P., Lukas, J., Fry, A. M., Bartek, J., and Nigg, E. A. (1999) Nat. Cell Biol. 1, 88-93[CrossRef][Medline] [Order article via Infotrieve] |
39. | Spruck, C. H., Won, K. A., and Reed, S. I. (1999) Nature 401, 297-300[CrossRef][Medline] [Order article via Infotrieve] |
40. |
Hinchcliffe, E. H.,
Li, C.,
Thompson, E. A.,
Maller, J. L.,
and Sluder, G.
(1999)
Science
283,
851-854 |
41. | Matsumoto, Y., Hayashi, K., and Nishida, E. (1999) Curr. Biol. 9, 429-432[CrossRef][Medline] [Order article via Infotrieve] |
42. |
Lacey, K. R.,
Jackson, P. K.,
and Stearns, T.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
2817-2822 |
43. | Brown, C. R., Doxsey, S. J., White, E., and Welch, W. J. (1994) J. Cell. Physiol. 160, 47-60[Medline] [Order article via Infotrieve] |
44. | Fukasawa, K., Wiener, F., Vande Woude, G. F., and Mai, S. (1997) Oncogene 15, 1295-1302[CrossRef][Medline] [Order article via Infotrieve] |
45. | Chiba, S., Okuda, M., Mussman, J. G., and Fukasawa, K. (2000) Exp. Cell Res. 258, 310-321[CrossRef][Medline] [Order article via Infotrieve] |
46. | Mussman, J. G., Horn, H. F., Carroll, P. E., Okuda, M., Tarapore, P., Donehower, L. A., and Fukasawa, K. (2000) Oncogene 19, 1635-1646[CrossRef][Medline] [Order article via Infotrieve] |
47. | Fukasawa, K., Choi, T., Kuriyama, R., Rulong, S., and Vande Woude, G. F. (1996) Science 271, 1744-1747[Abstract] |
48. | Morris, V. B., Brammall, J., Noble, J., and Reddel, R. (2000) Exp. Cell Res. 256, 122-130[CrossRef][Medline] [Order article via Infotrieve] |
49. |
Oren, M.
(1999)
J. Biol. Chem.
274,
36031-36034 |
50. | Tarapore, P., and Fukasawa, K. (2000) Cancer Invest. 18, 148-155[Medline] [Order article via Infotrieve] |