[CANCER RESEARCH 58. 396-401.
February 1. 1998)
Advances in Brief
p53 and pRb Prevent Rereplication in Response to Microtubule Inhibitors by
Mediating a Reversible G! Arrest
Shireen Hussain Khan and Geoffrey M. Wahl1
Gene Expressiint Lah. The Salk Institute ¡G.M. W., S. H. K.I, and Department of Biology, University of California at San Diego, La Jolla. California 92037 ¡S.H. K.I
Abstract
Materials and Methods
Cell cycle checkpoints are safeguards that ensure the initiation of
downstream events only after completion of upstream processes. The
tumor suppressors p53 and pRb prevent initiation of a second round of
replication in response to spindle inhibitors, but it has yet to be proven
that this is a mitotic checkpoint response. We show that asynchronous
human fibroblasts arrest in (., with 4 N DNA content after nocodazole
treatment, whereas isogenic p53- and pRb-defìcient fìbroblasts rerepli
cate. Importantly, nocodazole elicits a reversible arrest in G0-G, synchro
nized normal human fibroblasts but not in isogenic p53-deficient deriva
tives. Furthermore, the G, cyclin-dependent kinase inhibitors p21 and pl6
Cell Culture. WS Ineo, WS1E6, and WS1E7 human fibroblasts were pre
pared (3) and maintained as described previously (12). p53+/- and p53-/MEFs- were isolated from transgenic mouse embryos (13). p21+/and
p21—/—breeding pairs were kindly provided by Dr. Philip Leder (Harvard
Medical School. Boston, MA). p21+/— and p21—/—MEFs were isolated
from transgenic mouse embryos 13 days after conception. pl6—/—MEFs were
also play critical roles in limiting rereplication. Hence, p53 and pRb are
required during (., to prevent entry into a replicative cycle and appear to
provide a connection between the structural integrity of the microtubules
and the cell cycle machinery in interphase cells.
serum. All other MEFs were maintained in DMEM containing 10% dialyzed
fetal bovine serum.
Genotyping p21+/- and p21—/—
MEFs. Early-passage fibroblasts were
freshly isolated from embryos generated by a cross of p21 —¿/—
and p21 +/—
mice. Genotypes of p21-/and p21+/MEFs were determined by PCR
Introduction
analysis of genomic DNA. The p21 gene was targeted for disruption by a
construct containing a neomycin resistance cassette flanked by p21 genomic
sequences (14). The neomycin genes were amplified using the primers (5'AGAGCCTATTCGGCTATGACTG-3')
and (5'-TTCGTCCAGATCATCCTGATG-3'). Wild-type p21 was detected using the primers (5'-CCGTGGACAGTGAGCAGTTG-3')
and (5'-GCAGCAGGGCAGAGGAAGTA-3').
The
conditions for PCR amplification were 30 cycles of 94°Cfor 20 s, 60°Cfor
30 s, and 72°Cfor l min. All PCR reactions were repeated in triplicate to
Aneuploidy occurs in a high proportion of tumors, suggesting that
the mechanisms that ensure faithful chromosome segregation are
often compromised during cancer progression. Recent data suggest
that two genes frequently mutated in human cancer, p53 and pRb, play
important roles in limiting aneuploidy in human and rodent cells.
Thus, cells lacking functional p53 or Rb initiate a second round of
DNA replication without cytokinesis, a process we will refer to as
rereplication. The propensity of p53-deficient fibroblasts to rereplicate
(1-3) and to become polycentrosomal (4) probably contributes to the
observed increase in aneuploidy. Spindle inhibitors also induce rerep
lication in budding yeast spindle checkpoint mutants, which have
defects in BUB genes (5). This led to the proposal that p53 is involved
in the mammalian analogue of this mitotic checkpoint (1). However,
two observations suggest that the defects leading to rereplication in
p53-deficient mammalian cells may not be due to a defect in the
spindle assembly checkpoint: (a) wild-type rodent cells treated with
nocodazole enter and then exit mitosis, after which p53 induction
occurs (2). This is in contrast to wild-type yeast strains, which remain
arrested in mitosis and exhibit elevated p34cdt2/CDC21ikinase activity
throughout nocodazole treatment (5); and (b) the propensity of pRbdeficient fibroblasts to rereplicate due to a defect in mitosis is unex
pected because the majority of data indicate that it regulates progres
sion from G | into S phase (6, 7). To reconcile these inconsistencies,
the studies described here were designed to test the hypothesis that
rereplication is a consequence of "mitotic slippage," a process by
which cells gradually escape the mitotic block induced by spindle
inhibitors and enter a G,-like state (2, 8-11). Our data support this
proposal and elucidate some of the cell cycle regulatory pathways
involved in this response.
Received 11/14/97; accepted 12/19/97.
The costs of publication of this article were defrayed in pan by the payment of page
charges. This article must therefore be hereby marked atlrertisemenl in accordance with
18 U.S.C. Section 1734 solely to indicale this faci.
1To whom requests for reprints should be addressed.
kindly provided by Dr. Manuel Serrano (Centro Nacional de Biotecnologia.
Madrid, Spain). pl07+/-;pl30-rVand pl07-/-;pl30-/MEFs were
kindly provided by Dr. David Cobrinik (Columbia University, New York,
NY). pl6—/—MEFs were maintained in DMEM containing 10% fetal bovine
confirm results.
Cell Cycle Analysis. Asynchronous cultures were split 1:3 or 1:4 into
media with or without nocodazole (0.05-0.1 /ng/ml) or with 0.5 /Ag/ml Colcemid. PI staining and FACS analysis was done as described previously (3). To
determine whether the nocoda/.ole arrest was reversible, serum-arrested cells
were treated with nocodazole for 48 h, washed with PBS —¿
, and fresh medium
containing BrdUrd was added for 24-48 h. Sample preparation and analysis of
continuously labeled BrdUrd cells were conducted as described previously
(12). For PI analysis, cells with greater than 4 N DNA content were quantitated
by gating events to the right of the 4 N peak on histogram plots. For BrdUrd
analysis, the percentage of nocodazole-treated or nocodazole-released
cells
entering S phase was determined by taking the sum of the BrdUrd-positive
populations.
MPM-2/PI Bivariate Flow Cytometry. Cells were grown to -80% con
fluence, and asynchronous cultures were split 1:3 or 1:4 into medium with or
without 0.05 /ig/ml nocodazole. MPM-2 staining was done according to
published procedures with minor modifications (2. II). At specified time
points, adherent cells were trypsinized. pooled with floating cells, and fixed in
1% paratbrmaldehyde for 10 min at room temperature. After staining with
MPM-2 (Upstate Biotechnology. Lake Placid, NY) and goat anti-mouse-FITC
(Boehringer Mannheim. Mannheim. Germany), the cells were washed and
resuspended in 5 fig/ml PI containing 200 fig/ml RNase. Samples were
analyzed on a Becton Dickinson FACScan, and data analyses were completed
using CellQuest software.
Imimi Molilo! Analysis. Asynchronous or 0,,-G, synchronized cultures of
WS 1neo and WS1E6 were treated with and without 0.05 ng/ml nocodazole for
24 and 48 h. Lysates were prepared and quantitated as described previously
(12). Fifty (50) fig of protein were resolved on a 6.5 or 10% SDS polyacryl2 The abbreviations used are: MEF. mouse embryonic fibroblasc PI. propidium iodide:
FACS, fluorescence-activated
cell sorter; BrdUrd. bromodeoxyuridine;
CDK. cyclindependent kinase.
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SPINDLE
amide gel and transferred onto nitrocellulose membranes
Schuell. Keene, NH). Membranes were probed with mouse
bodies anti-pRb 3C8 (Canji, San Diego, CA). anti-21 (Santa
ogy. Santa Cruz. CA). anti-cyclin Bl (PharMingen, San
anti-ß-actin (Sigma Chemical Co., St. Louis, MO). The
treated with a secondary sheep anti-mouse-HRP antibody
INHIBITORS
INDUCE
(Schleicher and
monoclonal anti
Cruz Biotechnol
Diego. CA). and
membranes were
for I h at room
temperature. Detection was done by ECL chemiluminescence reagents (Du
pont NEN, Boston, MA) according to the manufacturer's protocol.
A G, ARREST
Untreated
36h
Nocodazole
36h
o
0)
Results and Discussion
Nocodazole Induces a Transient Arrest in Mitosis in WildType, p53-deficient, and pRb-deficient Cells. It has been proposed
that p53 is required to activate the spindle assembly checkpoint in
mammalian cells (1). This model proposes that p53 induces cell cycle
arrest if the spindle is not assembled properly. A central requirement
of this model is that mutation of any gene involved in the p53 signal
transduction pathway should relieve the dependency of progression
through mitosis on completion of spindle assembly. If p53 and pRb
participate in a spindle checkpoint, cells defective for either gene
product should fail to arrest in mitosis, enter a succeeding replicative
cycle without delay, and rereplicate in the presence of spindle inhib
itors. In contrast, cells with an intact spindle checkpoint should arrest
in mitosis.
We tested the checkpoint model by determining the kinetics of
entry into and progression through M phase and the timing of rereplication in normal (WS 1neo), p53-deficient (WS1E6), and pRb-deficient (WS1E7) fibroblasts challenged with nocodazole. The E6 and
E7 oncoproteins from HPV 16 may have activities in addition to
inhibiting p53 or pRb functions (15); however, the WS1E6 and
WS1E7 modified human fibroblasts exhibit similar responses to no
codazole as p53—/—and Rb—/—MEFs (3). Therefore, differences in
nocodazole responsiveness in these cells most likely relate to the
inactivation of p53 and pRb.
Flow cytometry was used to quantify cells in M phase by simulta
neously scoring for the mitotic protein marker MPM-2 and DNA
content using PI (Fig. IA). MPM-2 recognizes a conserved epitope
shared by mitotic phosphoproteins: the appearance of the MPM-2
antigens correlates with entry into mitosis, whereas the dephosphorylation of these antigens correlates with anaphase onset (16). Hence,
cells exhibiting both 4 N DNA content and MPM-2 reactivity should
be in M phase. Nocodazole-treated cells with greater than 4 N DNA
content are inferred to have rereplicated without completing nuclear
kinesis.
Exponential cultures of WS 1neo, WS1E6, and WS1E7 challenged
with 0.05 ju,g/ml nocodazole all displayed a peak of MPM-2 reactivity
after 24 h of drug exposure (Fig. Iß).Such a peak is not observed in
untreated populations due to the rapid turnover of cells from metaphase into anaphase in exponential populations. The MPM-2 signal
gradually decreased with similar kinetics for each cell type after 36,
48, and 72 h (Fig. IB). The peak and subsequent decline of the
MPM-2 signal suggests that each cell type enters, arrests, and then
exits mitosis. It is likely that a mitotic checkpoint mediates this
transient mitotic arrest during nocodazole treatment. However, the
transient arrest is evident in wild-type, p53-deficient and pRb-deficient fibroblasts, which suggests that p53 and pRb are not involved in
the mitotic checkpoint triggered by spindle inhibitors. Concurrent
with the decreasing MPM-2 signal, the percentage of WS1E6 and
WS1E7 cells with greater than 4 N DNA content began to increase
between 24 and 36 h of nocodazole exposure (Fig. 1C). These results
show that, irrespective of p53 or pRb status, the cells pass through an
MPM-2-positive state before presumably entering a phase similar to
G, with 4 N DNA content. When the cells enter this G,-like state, they
no longer express the MPM-2 antigen (Fig. Iß).During prolonged
DNA Content
B
WSIneo Uni
WS 1neo Noe
WS1E6Unt
WS1E6NOC
WS1E7Unl
WS1E7NOC
36
Time (h)
Fig. I. Asynchronous WSIneo, WS1E6. and WSIE7 cultures treated with nocodazole
enter and exit mitosis with similar kinetics. Asynchronous cultures of WSIneo. WS1E6.
and WSIE7 were treated with or without 0.05 fig/ml nocodazole and fixed at the specified
lime points. The cells were stained with primary MPM-2 monoclonal antibodies followed
by secondary goat anti-mouse-FITC antibodies. The cells were stained with 5 ^.g/ml PI
prior to FACS analysis. This study was repeated a minimum of three times, and repre
sentative data are shown. A. dot plots of WS I neo treated w ith and without nocodazole for
36 h. Events in the upper right (¡itailranlwere gated out as MPM-2-positive cells. B. line
graphs of MPM-2 positivity in WS 1neo, WS l E6. and WS 1E7 over a 72-h time course. O
line graphs of percentage of cells with >4 N DNA content over a 72-h time course. Cells
with more than 4 N DNA content were quantitated by gating events to the right of the 4
N peaks on histogram plots.
nocodazole treatment, the wild-type cells remain arrested in this
presumed 4 N G,. whereas the p53- and pRb-deficient fibroblasts
initiate a second round of replication.
Thompson and colleagues (2) proposed that murine prolympho
cytes enter G, by default after sustained nocodazole treatment and
undergo p53-mediated apoptosis. Our MPM-2 analyses of human
fibroblasts are compatible with their proposal that cells can slowly
progress into G[ without completion of mitosis or cytokinesis, but as
shown below, human fibroblasts arrest reversibly rather than undergo
cell death. This discrepancy in the nocodazole-induced G, response
may be due to differences in species and/or cell types. Hence, it is
397
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SPINDLE
WS1 neo
Time (h)
Nocodazole
24
24
-
+
48
-+-
INHIBITORS
INDUCE A O, ARRKST
The CDK Inhibitors p21 and pl6 Are Components of the
Arrest Pathway Activated by Spindle Inhibitors. p53 can maintain
pRb in a hypophosphorylated state through induction of p21 (25),
leading to the reasonable expectation that p21 may be partly necessary
for the p53-mediated arrest triggered by spindle inhibitors. We iso
lated p21 -/- and p21 +/- MEFs (see "Materials and Methods") and
WS1E6
48
24
24
48
48
+
-
+
Fig. 2. Hypophosphorylated pRh accumulation in WS I neo cells but noi in WSIIÌ6
cells treated with nocodu/.ole. Lysates were prepared from asynchronous cultures of
WS I neo and WS I lift treated with and without 0.05 fig/ml nocoda/ole for 24 and 48 h.
Fifty /ig of prolein \vere loaded in each lane. After Western transfer, the membrane was
probed for pRb. The slower migrating band corresponds to the hyperphosphorylated forni
of pRh. and the faster migrating hand corresponds to hypophosphorylated pRb.
determined their ability to arrest in response to prolonged Colcemid
treatments. The spindle inhibitor Colcemid was used because pro
longed exposure to nocodazole appeared to be toxic to these cells.
p21+/— MEFs treated with 0.5 ju.g/ml Colcemid arrested primarily
with 4 N DNA content and did not show a significant increase in the
polyploid population (12%) compared with the untreated control (9%;
data not shown). Whereas an average of 13% of untreated p21 —¿I—
MEFs were >4 N, a 48-h Colcemid treatment resulted in an average
of 25% cells >4 N in three separate experiments (Fig. 3C). These
results indicate that the arrest induced by spindle inhibitors should be
mediated by activation of, or increase in, the intracellular p21 pool.
Western blot analysis showed that the p21 levels were 6-fold higher in
nocodazole-treated WS 1neo lysates compared with the untreated con
trol (data not shown). These results indicate that the signal inducing a
G, arrest in response to nocodazole is achieved through a p53dependent increase in the p21 protein level. A prior analysis showed
that fibroblasts derived from p21-deficient mice did not rereplicate
when exposed to Colcemid for 24 h ( 14); however, it is possible that
likely that in human cells. p53 and pRb mediate cell cycle exit from
a 4 N G|-like state in response to antimicrotuhule agents.
pRb Is Hyperphosphorylated
in p53-deficient Human Cells
Treated with Nocodazole. pRb is converted to a hyperphosphoryluted form that fails to bind E2F family members around the G,
restriction point (7) and is then converted back to a hypophosphory
lated inhibitory form at the metaphase-anaphase transition (I7). If the
decline in MPM-2 signal results from progression of M-phase cells
into G,, then pRb should largely be in the hypophosphorylated form.
Hence, normal fibroblasts should exhibit hypophosphorylated pRb
during prolonged treatment with spindle inhibitors, whereas p53deficient fibroblasts should accumulate hyperphosphorylated pRb.
Immunoblot analysis of pRb phosphorylation was done by prepar
ing lysates from asynchronous cultures of untreated and nocodazoletreated WS Ineo and WS IE6 fibroblasts. WS Ineo cells treated with
0.05 fxg/ml nocodazole for 24 h showed an increase in the amount of
hypophosphorylated pRb compared with the untreated control (Fig.
2). After 48 h in nocoda/ole. hyperphosphorylated pRb was undetectable in WS Ineo lysates. and hypophosphorylated pRb was the pre
dominant species. In contrast, the amount of hyperphosphorylated
pRb in WSIE6 lysates treated with nocoda/ole was similar to the
untreated controls at early and late time points (Fig. 2). These bio
chemical results are compatible with the proposal that nocoda/ole
treatment can allow progression of normal cells into a 4 N G,-like
state. The presence of abundant hyperphosphorylated pRb in p53deficient cells after 24-48 h of nocodazole treatment is consistent
with their ability to enter S-phase during this time interval.
Untreated
Colcemid
B
We have shown previously that WS1E7 human fibroblasts typically
have a higher percentage of >4 N cells after exposure to nocoda/ole
than Rb-/MEFs (3). This raised the possibility that other targets of
the HPV 16 E7 oncoprotein. such as the pRb-related proteins p 107
and p 130, may be involved in preventing rerepl¡cation. After 48 h of
nocodazole treatment. pl07-/-;pl30-/double-knockout MEFs
did not show significant increases in the >4 N populations relative to
wild-type or pl07+/-;pl30+/MEFs (data not shown). These
results indicate that, among this related group of pocket proteins. pRb
is the most significant regulator of downstream processes that lead to
rereplication.
pRb negatively regulates the ability of E2F-1, E2F-2, and E2F-3 to
function as transcriptional regulators (7). E2F-1 activates the tran
scription of several genes required for DNA synthesis, such as DHFR
(18), and the human ORCI gene (HsOrd; Ref. 19). ORCI is one
component of the budding yeast origin recognition complex that is
required for initiation of replication (20, 21 ). Therefore, it is conceiv
able that the p53 pathway may prevent rereplication by activation of
a CDK inhibitor that maintains pRb in the hypophosphorylated form
thai binds E2Fs (22-24). This would prevent E2F-1 from activating
HsOrcl or other genes that may be essential for origin licensing and
initiation of DNA replication. Studies testing this hypothesis are in
progress.
Fig. 3. p2l and plfi null MFFs rereplicate in response to Colcemid. Asynchronous
cultures of p53+/-. p53-/-.
and p21 -/- MEFs were split into media with or without
0.5 fig/ml Colcemid. After 48 h. the cells were fixed and stained w ith 20 fig/ml PI for flow
cylometric analyses. pl6-/MEFs were processedby the same method hut in a separate
experiment. These studies were repeated a minimum of three times, and representative
data are presented. Histogram plots of untreated or Coleemid-treated MEFs after 48 h are
illustrated in A (p53+/- MEFs). B (p53-/MEFs). C (p2\-l- MEFs), and D (pl6-/MEFs).
398
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SPINDLE
Fig. 4. Nocodazole induces a p53-dependent arresi in G,,-G, synchro
nised cells. WS Ineo and WSIE6 cells were synchronized in GC>-G, by
serum deprivation for 48 h. The cells were released into complete media
with or without 0.05 /ig/ml nocodazole and with or without Brdllrd. At the
specified time points, cells were either fixed in ethanol and processed for
anti-BrdUrd-FITC and PI analysis, or lysales were prepared. A. percentage
of WSIneo (•)and WS1E6 (D) cells that are BrdUrd positive in the
presence of nocodazole over a 72-h time course. The percentages were
obtained by averaging the results of three independent experiments. B,
Western blot analysis of cyclin Bl levels in G,,-G| synchronized WSIneo
and WS1E6 lysates at 24 and 48 h with and without nocodazole. The
corresponding ß-actinlevels are shown as a loading control. C. percentage
of BrdUrd positive cells after 24 h (•)and 48 h (D) of release into complete
media. Nocoda/.ole-induced G, arrest is reversible. To determine revers
ibility. G0-Gj synchronized cells were treated with nocodazole (M>r) for
48 h and released into complete media for 24 and 48 h. This release was
compared with that of cells that were serum starved (.v.v)for 48 h prior to
release into complete media.
INHIBITORS
INDUCE A G, ARREST
36
48
72
Time (h) in Nocodazole
B
WSIneo
Time (h)
Nocodazole
0
24 24 48 48
WSIneo
Noe rotease
the time used in these studies contributed to the negative result. Our
data implicate p21 function in the signal transduction pathway acti
vated by spindle inhibitors.
Over 40% of p53-/MEFs (Fig. 3ß)and more that 50% of
pRb-/MEFs (3) became polyploid after 48 h of exposure to spindle
inhibitors, which is significantly greater than the maximum percent
age (30%) of polyploid cells observed in p21-/MEFs (Fig. 3C).
This raised the possibility that additional CDK inhibitors might be
activated upon exposure to microtubule inhibitors. Because p 16 can
regulate pRb phosphorylation by inhibiting CDK4-6/cyclin D kinase
activity (26). we compared the ability of pl6—/—and pl6+/+ MEFs
to rereplicate during Colcemid treatment. Untreated populations of
pl6+/+ MEFs started with fewer than 15% polyploid cells (data not
shown), and untreated pl6—/—MEFs had approximately 10-15%
cells >4 N (Fig. 3D). pl6+/+
MEFs did not exhibit a significant
increase in the number of cells with >4 N DNA content after 48 h of
Colcemid treatment (Fig. 3A and data not shown), whereas 30-35%
of pl6-/MEFs became >4 N under the same conditions (Fig. 3D).
Because pl6 and p53 are thought to operate in separate pathways (27).
pi6 deficiency may allow rereplication due to accumulation of hyperphosphorylated pRb.
Our data indicate that pi6 cannot complement p21 deficiency, and
that p21 cannot complement pl6 deficiency to prevent rereplication.
WS1E6
0
24 24
48 48
WS1E6
ss release
WS1E6
Noe release
This suggests that these two CDK inhibitors prevent rereplication by
involvement in nonoverlapping regulatory pathways that eventually
converge at pRb. Furthermore, the data indicate that loss of either p21
or pl6 enables rereplication to occur but at a lower rate than that
achieved by inactivation of either p53 or pRb.
The Nocodazole-induced Arrest Does Not Require Progression
through the Cell Cycle. It has been assumed that the signal for arrest
is generated after cells replicate their DNA and enter a 4 N G,.
However, another possibility is that the signal can be generated in
cells that have not yet attempted DNA replication. We assessed
whether nocodazole would activate a p53-dependent arrest in G,,-G|
synchronized cells. WSIneo and WS1E6 cultures were synchronized
by serum depletion for 48 h followed by release into serum, nocoda
zole, and BrdUrd to monitor S-phase progression. The number of cells
that incorporate BrdUrd during nocoda/ole exposure was measured
over the ensuing 72 h. In three independent experiments, only 8-20%
of WSIneo cells treated with nocodazole were BrdUrd positive over
a 72-h time course (Fig. 4A). By contrast, 60-80% of WS1E6
populations incorporated BrdUrd under the same conditions (Fig. 4A).
G0-G,-synchronized WS1E7 fibroblasts also readily entered S phase
when released into nocodazole (data not shown). Interestingly,
WS1E6 populations did not enter the cycle when treated with high
concentrations of nocodazole (>50() nM). suggesting that a p53-
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1998 American Association for Cancer Research.
SPINDLE
INHIBITORS
INDUCE A O, ARREST
independent mechanism prevents cell cycle entry at high drug con
centrations (data not shown). These data suggest that low concentra
tions of nocodazole disrupt a structure or process in G0-G, cells that
elicits an arrest through activation of p53- and pRb-mediated signal
transduction pathway(s).
Cyclin Bl Western analysis provided further evidence that the
arrest signal is transduced in cells that have not entered the cycle.
Cyclin Bl accumulates as cells enter mitosis and is subsequently
degraded as cells enter G, (28, 29). G0-G,-synchronized WS 1neo
fibroblasts released for 48 h into nocodazole accumulate little, if any,
cyclin Bl, whereas WS1E6 populations show substantial levels of
cyclin Bl under the same conditions (Fig. 4B). WS1E6 lysates display
a sharp decline in the cyclin B l levels at 48 h. which is consistent with
the cell cycle and MPM-2 analyses (Fig. l, B and C). indicating that
these cells enter and exit mitosis and progress into G, without divid
ing. Taken together, these data indicate that entry into the cell cycle is
not required for activation of the G, arrest pathway triggered by
antimicrotubule agents.
Reversibility of the Nocodazole-induced G, Arrest. One of the
requirements of a checkpoint is that the arrest should reverse upon
removal of the Stressor. The reversibility of the nocodazole-induced
arrest was tested by releasing G,,-arrested WS 1neo fibroblasts into
complete media with nocodazole and BrdUrd for 48 h, followed by
release into complete media containing BrdUrd. Approximately 50%
of untreated WS 1neo populations entered S phase 24 h after release
into complete media, and ~78% were BrdUrd positive after 48 h (Fig.
4C). Similar results were obtained in G„-arrested
cells incubated for
48 h in nocodazole prior to release in drug-free media; 42 and 70% of
WS 1neo cells were BrdUrd positive after 24 and 48 h, respectively
(Fig. 4C). These data show that the reversibility of the p53-dependent
G, arrest triggered by nocodazole is comparable with that observed
for ribonucleotide depletion (12).
The data presented above reveal that the arrest triggered by
microtubule-destabilizing agents fulfills most of the criteria for a
cell cycle checkpoint (30). Some important requirements for a
checkpoint are: (a) mutations in any component of the checkpoint
pathway prevent cell cycle arrest during exposure to the conditions
that provoke arrest in the wild-type cells; (b) genetic instability is
increased and viability is decreased in the mutants compared with
the wild-type cells; and (c) checkpoint-induced arrest is reversible.
Clearly, DNA replication is no longer dependent on completion of
mitosis in p53- and pRb-deficient cells challenged with nocoda
zole. Genetic instability is increased, as demonstrated by the
nocodazole-induced increases in ploidy of cells with defects in the
tumor suppressors p53 and pRb or the CDK inhibitors p21 and p 16.
Our data also show that the G0-G, arrest induced by nocodazole is
largely reversible. However, contrary to observations made in
some yeast checkpoint mutants (31), the viability of p53-deficient
murine cells is greater than that of wild-type cells after nocodazole
treatment due to a reduced frequency of apoptosis (2). Taken
together, the data lead us to propose that p53 is in a G, checkpoint
that responds to the consequences of microtubule disruption and
prevents advancement into the cell cycle during challenge with
microtubule inhibitors.
Although the signal that activates the p53- and pRb-dependent
G| checkpoint remains to be determined, our data make two
previously proposed candidates less likely: (a) hyperploidy may
elicit an arrest signal, because the resulting G, cells would have a
4 N DNA content. However, our ability to induce an arrest in G()
cells indicates that the signal can be elicited in cells that have a 2
N DNA content; and (b) DNA damage might be incurred as a result
of spindle disruption, and this might trigger a p53-dependent G,
arrest or apoptosis (2). DNA damage is an unlikely signal, because
we showed that the arrest can be initiated in G,,-arrested cells, and
nocodazole is not likely to cause DNA damage in a noncycling
cell. Furthermore, we showed previously that DNA damage in
duces a permanent arrest resembling senescence in human fibro
blasts (32, 33); yet the arrest induced by nocodazole was readily
reversed upon drug removal. Finally, though cells from patients
with ataxia telangiectasia have a defective p53-mediated response
to DNA damage (34), they do not rereplicate during nocodazole
challenge (data not shown). Thus, we propose that the arrest
triggered by microtubule-destabilizing agents constitutes a second
DNA damage-independent, p53-mediated G, checkpoint.
It is tempting to propose that disruption of the microtubule network
itself may elicit an arrest signal. Taxol, a microtubule-stabilizing
agent, induced a G, arrest in G0-G, synchronized, nontransformed
mammalian fibroblasts, whereas SV40 T antigen-transformed cells
did not arrest (35). Although taxol and nocodazole work antagonisti
cally, the data imply that the microtubule organization and/or dynam
ics may affect progression through G, and into S phase. Indeed,
interphase cells rely on microtubules to establish polarity and to
mediate vesicle transport between organdÃ-essuch as the endoplasmic
reticulum and the Golgi apparatus (36). Further studies are in progress
to determine the extent and consequences of microtubule damage in
interphase cells and how this might be signaled to the cell cycle
machinery.
p53 was previously implicated in G, arrest responses triggered
by DNA damage, ribonucleotide pool depletion, and overexpression of oncogenenes that activate the mitogen-activated protein
kinase (MAPK) pathway (27, 37). Interestingly, overexpression of
mos, a potent activator of the MAPK pathway, induces p53+/+
MEFs to arrest in a 4 N G,-like state (37), similar to the arrest we
observe after nocodazole treatment. It is possible that oncogenic
activation of the MAPK pathway elicits a similar change in
microtubule-dependent structures as that produced by nocodazole
treatment of cells in G,. We show here that p53 mediates a G,
checkpoint activated by microtubule inhibitors. Thus, absence of a
functional p53 pathway removes many of the safeguards that
prevent accumulation of genetic abnormalities, resulting in cells
that may acquire changes in chromosome content when exposed to
the appropriate stressing and selective conditions. Importantly,
agents that interfere with microtubule dynamics are among the
most commonly used in cancer treatment (38). However, the high
frequency of defects in the p53 pathway in the most common
human neoplasms raises the possibility that treatments using these
agents may increase genomic instability and accelerate the rate of
emergence of genetic variants.
Acknowledgments
We thank Dr. Aldo Di Leonardo for thoughtful discussions and Dr. Yan Ren
for technical assistance with isolation of p21-deficient MEFs.
References
1. Cross, S. M. Sanchez, C. A.. Morgan. C. A., Schimke, M. K., Ramel. S., Idzerda.
R. L.. Raskind, W. H.. and Reid. B. J. A p53-dependenl mouse spindle checkpoint.
Science (Washington DC), 267: 1353-1356, 1995.
2. Minn. A. J.. Boise. L. H., and Thompson. C. B. Expression of Bcl-xL and loss of p53
can cooperate to overcome a cell cycle checkpoint induced by mitotic spindle
damage. Genes Dev., 10: 2621-2631. 1996.
3. Di Leonardo. A., Hussain-Khan, S.. Linke, S. P.. Greco, V., Seidita. G., and Wahl, G. M.
DNA rereplication in the presence of mitotic spindle inhibitors in human and mouse
fibroblasts lacking either p53 or pRb function. Cancer Res.. 57: 1013-1019, 1997.
4. Fukasawa. K.. Choi, T.. Kuriyama. R.. Rulong. S.. and Vande Woude. G. F. Abnor
mal centrosome amplification in the absence of p53. Science (Washington DC), 271:
1744-1747, 1996.
5. Hoyt, M. A.. Totis, L., and Roberts. B. T. S. cenrisiae genes required for cell cycle
arrest in response to loss of microtubule function. Cell. 66: 507-517, 1991.
6. Goodrich. D. W., Wang. N. P.. Qian, Y. W., Lee, E. Y., and Lee. W. H. The
400
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1998 American Association for Cancer Research.
SPINDLE
1.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
INHIBITORS
INDUCE A O, ARRI-ST
retinoblastoma gene product regulates progression through the GI phase of the cell
cycle. Cell. 67: 293-302, 1991.
Weinberg. R. A. The retinoblastoma protein and cell cycle control. Cell. 81: 323-330.
1995.
Charnla. Y.. Roumy. M., Lassegues, M., and Battin. J. Altered sensitivity to cholchicine and PHA in human cultured cells. Hum. Genet.. 53: 249-253, 1980.
Rung. A. L., Sherwood. S. W., and Schimke, R. T. Cell line-specific differences in the
control of cell cycle progression in the absence of mitosis. Proc. Nati. Acad. Sci.
USA, 87: 9553-9557, 1990.
Rieder. C. L., and Palazzo, R. E. Colcemid and the mitotic cycle. J. Cell Sci., 702:
387-392, 1992.
Andreassen. P. R., and Margolis. R. L. Microlubule dependency of p34cdc2 inactivation and mitotic exit in mammalian cells. J. Cell Biol., 127: 789-802. 1994.
Linke, S. P.. Clarkin. K. C. Di Leonardo, A., Tsou. A., and Wahl, G. M. A reversible
p53-dependenl GO/GI cell cycle arrest induced by ribonucleotide depletion in the
absence of detectable DNA damage. Genes Dev., 10: 934-947. 1996.
Huang, L-c.. Clarkin. K. C. and Wahl. G. M. p53 dependent cell cycle arrests are
preserved in DNA-activated protein kinase-deficient mouse fibroblasts. Cancer Res..
56: 2940-2944. 1996.
Deng, C.. Zhang, P.. Harper. J. W.. Elledge. S. J.. and Leder. P. Mice lacking
p21CIPI/WAFl
undergo normal development, but are defective in Gl checkpoint
control. Cell. 82: 675-684, 1995.
Tommasino. M.. and Crawford. L. Human papillomavirus E6 and E7: proteins which
deregulate the cell cycle. Bioessays, 17: 509-518. 1995.
Vandre, D. D.. and Borisy. G. G. Anaphase onset and dephosphorylation of mitotic
phosphoproteins occur concomitantly. J. Cell Sci.. 94: 245-258. 1989.
Ludlow, J. W.. Glendening. C. I... Livingston, D. M.. and DeCaprio, J. A. Specific
en/.ymatic dephosphorylation of the retinoblastoma protein. Mol, Cell. Biol.. 13:
367-372, 1993.
Blake. M. C., and A/izkhan, J. C. Transcription factor E2F is required for efficient
expression ot the hamster dihydrofolate reducÃ-asegene in vilro and in vim. Mol. Cell.
Biol.. 9: 4994-5002. 1989.
Ohtani, K.. DeGregori, J., Leone. G., Herendeen, D. R., Kelly. T. J., and Nevins,
J. R. Expression of the HsOrcI gene, a human ORCI homolog, is regulated by cell
proliferation via the E2F transcription factor. Mol. Cell. Biol., 16: 6977-6984.
1996.
Bell. S. P.. Kobayashi. R.. and Stillman. B. Yeast origin recognition complex
functions in transcription silencing and DNA replication (see comments]. Science
(Washington DC). 262: 1844-1849, 1993.
Micklem, G.. Rowley. A.. Harwood. J.. Nasmyth, K., and Diffley, J. F. Yeast origin
recognition complex is involved in DNA replication and transcriptional silencing.
Nature (Lond.), 366: 87-89, 1993.
Bagchi. S.. Weinmann. R., and Raychaudhuri, P. The retinoblastoma protein copurifies with E2F-I. an El A-regulated inhibitor of the transcription factor E2F. Cell. 65:
1063-1072. 1991.
Chellappan, S. P.. Hiebert. S., Mudryj, M.. Horowitz, J. M., and Nevins, J. R. The
E2F transcription factor is a cellular target for the RB protein. Cell. 65: 1053-1061.
1991.
24. Bandara. L. R.. and La. T. N. Adenovirus Eia prevents the retinohlasloma gene
product from complexing with a cellular transcription factor. Nature (Lond.), 351:
494-497, 1991.
25. EI-Deiry. W. S., Tokino. T.. Velculescu, V. E., Levy. D. B.. Parsons, R., Trent, J. M.,
Lin. D., Mercer. W. E.. Kinzler, K. W., and Vogelstein, B. WAF1. a potential
mediator of p53 tumor suppression. Cell, 75: 817-25. 1993.
26. Serrano, M.. Hannon. G. J.. and Beach, D. A new regulatory motif in cell cycle
control causing specific inhibition of cyclin D/CDK4. Nature (Lond.). J66: 704-707.
1993.
27. Serrano. M., Lin, A. W., McCurrach. M. E., Beach, D.. and Lowe. S. W. Oncogenic
ras provokes premature cell senescence associated with accumulation of p53 and
pl6INK4a. Cell. 88: 593-602. 1997.
28. Holloway, S. L.. Glotzer, M.. King. R. W.. and Murray. A. W. Anaphase is initiated
by proteolysis rather than by the inactivation of maturation-promoting factor. Cell,
73: 1393-1402, 1993.
29. Surana. U.. Amon, A., Dowzer, C., McGrew. J., Byers, B.. and Nasmyth. K.
Destruction of the CDC28/CLB mitolic kinase is not required for the metaphase to
anaphase transition in budding yeast. EMBO J.. 12: 1969-1978, 1993.
30. Hartwell. L. H., and Weinert. T. A. Checkpoints: controls that ensure the order of cell
cycle events. Science (Washington DC). 2-Õ6:629-634. 1989.
31. Weinert, T. A., and Hartwell. L. H. The RAIW gene controls the cell cycle response
to DNA damage in Sacchammn-es cerevisiae. Science (Washington DC). 241:
317-322, 1988.
32. Linke. S. P.. Clarkin, K. C., and Wahl. G. M. p53 mediates permanent arrest over
multiple cell cycles in response to gamma irradiation. Cancer Res., 57: 1171-1179,
1997.
33. Di Leonardo, A., Linke. S. P., Clarkin. K., and Wahl. G. M. DNA damage triggers a
prolonged p53-dependent Gl arrest and long-term induction of Cip I in normal hunuin
fibroblasts. Genes Dev.. 8: 2540-2551. 1994.
34. Kastan, M. B.. Zhan, Q.. EI-Deiry, W. S., Carrier, R. Jacks, T., Walsh. W. V..
Plunkett. B. S.. Vogelstein. B., and Fornace. A. J. A mammalian cell cycle checkpoint
pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell. 71:
587-597, 1992.
35. Trielli. M. O.. Andreassen. P. R.. Lacroix. F. B.. and Margolis, R. L. Differential
Taxol-dependent arrest of transformed and nonlransformed cells in the Gl phase of
the cell cycle, and specific-related mortality of transformed cells. J. Cell Biol., 135:
689-700. 1996.
36. Allen. R. D.. Weiss, D. G., Hayden, J. H., Brown. D. T.. Fujiwake. H., and Simpson.
M. Gliding movement of and bidirectional transport along single native microtubules
from squid axoplasm: evidence for an active role of microiubules in cytoplasmic
transport. J. Cell Biol.. 100: 1736-1752. 1985.
37. Fukasawa. K., and Vande Woude. G. F. Synergy between the mos/mitogen-activated
protein kinase pathway and loss of p53 function in transformation and chromosome
instability. Mol. Cell. Biol., 17: 506-518. 1997.
38. Rowinsky. E. K., and Donehower. R. C. The clinical pharmacology and use of
antimicrotubule agents in cancer chernotherapeutÃ-cs. Phannacol. Ther., 52: 35-84,
1991.
401
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p53 and pRb Prevent Rereplication in Response to Microtubule
Inhibitors by Mediating a Reversible G 1 Arrest
Shireen Hussain Khan and Geoffrey M. Wahl
Cancer Res 1998;58:396-401.
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