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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 4 (February 15), 1999:
pp. 1390-1398
p21cip-1/waf-1 Deficiency Causes Deformed Nuclear
Architecture, Centriole Overduplication, Polyploidy, and Relaxed
Microtubule Damage Checkpoints in Human Hematopoietic Cells
By
Charlie Mantel,
Stephen E. Braun,
Suzanna Reid,
Octavian Henegariu,
Lisa Liu,
Giao Hangoc, and
Hal E. Broxmeyer
From the Departments of Microbiology/Immunology, Medical and
Molecular Genetics, Medicine, and the Walther Oncology Center, Indiana
University School of Medicine, Indianapolis, IN; and the Walther Cancer
Institute, Indianapolis, IN.
 |
ABSTRACT |
A recent hypothesis suggests that tumor-specific killing by
radiation and chemotherapy agents is due to defects or loss of cell
cycle checkpoints. An important component of some checkpoints is
p53-dependent induction of p21cip-1/waf-1. Both p53 and p21
have been shown to be required for microtubule damage checkpoints in
mitosis and in G1 phase of the cell cycle and they thus help to
maintain genetic stability. We present here evidence that
p21cip-1/waf-1 deficiency relaxes the G1 phase
microtubule checkpoint that is activated by microtubule damage induced
with nocodazole. Reduced p21cip-1/waf-1
expression also results in gross nuclear abnormalities and centriole overduplication. p53 has already been implicated in centrosome regulation. Our findings further suggest that the p53/p21 axis is
involved in a checkpoint pathway that links the centriole/centrosome cycle and microtubule organization to the DNA replication cycle and
thus helps to maintain genomic integrity. The inability to efficiently
upregulate p21cip-1/waf-1 in p21cip-1/waf-1
antisense-expressing cells in response to microtubule damage could
uncouple the centrosome cycle from the DNA cycle and lead to nuclear
abnormalicies and polyploidy. A centrosome duplication checkpoint could
be a new target for novel chemotherapy strategies.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HEMATOPOIESIS depends on accurate
duplication and transfer of genetic information during cell
proliferation and differentiation, which requires the precise ordering
of cell cycle events. Proliferating hematopoietic cells, like most
other cells, achieve this coordination by using cell cycle
checkpoints.1,2 As the name implies, checkpoints monitor
events at critical points in the cycle and stop the progress of some
processes until other processes have been completed.
Mutations in some checkpoint molecules, like p53, greatly increase the
frequency of gene loss and amplification and contribute significantly
to the etiology and progression of human tumors.3,4 Checkpoint loss has recently been linked to tumor-specific killing by
chemotherapy agents.5
The tumor suppressor, p53, is highly implicated in human tumorigenesis.
p53 transactivates the expression of several proteins, including
p21cip-1/waf-1 (p21).6,7 p21 is a major
inhibitor of several key cell cycle regulating enzymes
(cyclin-dependent kinases)7 that are ultimately controlled
in hematopoietic cells by numerous exogenous cytokines and growth
factors. Together, p53 and p21 are important in several cell cycle
arresting checkpoints such as the DNA damage checkpoint,8 the mitotic spindle checkpoint,9,10 and microtubule (MT)
damage checkpoints.11
Our previous investigations of the molecular mechanisms of
hematopoietic growth-factor signaling focused on the process of growth
factor synergy.12 It was noted that p21 levels were
synergistically elevated when the human growth factor-dependent cell
line MO7e was synergistically stimulated to proliferate with the
combination of steel factor and granulocyte-macrophage
colony-stimulating factor (GM-CSF).12 Also,
retroviral-mediated gene transfer of the sense sequence of the p21 gene
into myeloid progenitor cells enhanced the proliferative capacity of
the progenitors in response to cytokine stimulation.13
During follow-up studies in which retroviral-mediated gene transfer of
antisense p21 sequence was used to reduce p21 expression in MO7e cells,
we observed that cells with antisense reduced p21 expression manifested
a remarkable change in nuclear morphology and polyploidy, a loss of MT
checkpoints, and centriole abnormalities. These results and their
potential implications are reported here.
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MATERIALS AND METHODS |
Cells, antibodies, and cDNA probes.
Parental MO7e cells used for retroviral infection were provided by
Genetics Institute (Cambridge, MA).14 MO7e
cells were maintained in RPMI 1640 medium with 20% fetal calf serum
(FCS) plus 100 U/mL GM-CSF (Immunex Corp, Seattle, WA) as
described.12 For experiments using G0/G1 synchronized
cells, factor-starvation was performed in medium without GM-CSF for 18 hours as described previously.14 After initial selection,
MO7e cells transduced with retroviral vector were constantly maintained
in this medium plus 0.4 mg/mL G418 (Sigma Chemicals Co, St Louis, MO).
All cells were routinely visualized microscopically after cytospin
(Shandon Southern Products/Miles, Inc, Elkhart, IN) preparation and
staining with Wright-Giemsa (Leukostat; Fisher Diagnostics, Pittsburgh, PA). Anti-WAF-1 (p21) monoclonal antibody was obtained from Oncogene Research Products (Cambridge, MA). WAF-1 probe was a gift from G. Adami (University of Illinois at Chicago, Chicago,
IL).7 Parental HCT 116 (p21 +/+) cells and one clonal
heterozygous (p21+/ ) deficient and two different clonal
homozygous (p21/) deficient cell lines were kind gifts
from Drs B. Vogelstein and T. Waldman (The Johns Hopkins Oncology
Center, Baltimore, MD).15
Retroviral vectors, transductions, and Northern blot analysis.
The human p21 sequence was generated by reverse
transcriptase-polymerase chain reaction (RT-PCR) from HL60
cells7 and subcloned into the retroviral vector,
LXSN.16 The LXSN and AS vectors were shuttle packaged into
amphotropic packaging cells17 and retroviral supernatant
from high-titer clones was used to transduce MO7e cells. Transduced
cells were selected in medium containing 0.4 mg/mL G418. Total RNA was
isolated, separated by electrophoresis, blotted onto nylon membranes,
and probed with radiolabeled p21 sequences.
Transmission election microscopy.
Transmission election microscopy was performed after fixing cells in
2% paraformaldehyde, 1% glutaraldehyde, 50 mmol/L phosphate buffer,
pH 7.3, and embedded in Spurrs Resin (Structure Probe, Inc, West
Chester, PA), and 100-nm sections were cut on a diatome. The sections were stained with uranyl acetate and lead citrate and
viewed on a Zeiss 10B transmission electron microscope
(Carl Zeiss, Inc, Thornwood, NY).
Western blot analysis.
Western analysis was performed on polyvinylidene difluoride
(PVDF) membranes (Millipore Corp, Bedford, MA) with
cellular proteins extracted and electrophoresed and transferred as
previously described.12 Anti-p21 antibodies bound to
protein bands were visualized with horseradish peroxidase-conjugated
goat antimouse IgG secondary antibody and enhanced chemiluminescence
photography (Amersham, Arlington Heights, IL), followed by digital
image scanning and quantitation.
Cell cycle analysis.
Cell cycle analysis was performed on synchronized cells or cells in log
phase growth after treatment with either nocodazole, colcemid (Sigma),
or diluent by staining the DNA with propidium iodide (Sigma) and
analyzing it with a Becton Dickinson (San Jose, CA) FACscan flow
cytometer. Laser light scatter was used to gate out dead or dying
cells. Cell cycle proportions were calculated using the modfit (Verity
Software House, Topsham, ME) computer program, with the model that
makes the fewest mathematical assumptions. Hereafter, G0/G1 proportion
refers to the proportion of cells with 2N DNA content and G2/M refers
to that with 4N DNA content, with the S phase proportion being
intermediate. All recorded events (after gating out dead cells and
doublets) greater than the highest 4N channel are considered polyploid events.
Fluorescence in situ hybridization (FISH) and immunofluorescence
staining.
Cells were applied to glass slides using cytospin preparations,
permeabilized with 0.05% sodium dodecyl sulfate (SDS), and then fixed
with ethanol. Cells were then denatured with 70% formamide at 75°C
and washed with ethanol. Denatured, digoxigenin-labeled DNA probe
specific for the centromeric region of human chromosome 7 or 1 (Oncor,
Inc, Gaithersburg, MD) and mouse anti- tubulin (Sigma) were added
and incubated at 37°C. Sheep anti-digoxigenin-fluorescein isothiocyanate (FITC) and horse antimouse-Texas Red (Oncor, Inc) were
then added for 15 minutes. Slides were washed with 0.1% tween 20 and
then with water and covered with 4,6-diamidino-2-phenylindole (DAPI) antifade solution and covered with glass slips.
They were examined and digitally recorded with a fluorescent microscope equipped with a cooled CCD camera and image acquisition and processing software (Vysis, Inc, Downers Grove, IL). Statistical tests for significance were performed using the Student's t-test.
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RESULTS |
To reduce p21 expression in MO7e cells, we generated the rectoviral
vector, L(ASp21)SN, containing full-length human antisense p21 sequence
and a selectable marker gene (neo). The antisense sequence is
transcriptionally regulated by the Moloney murine leukemia virus LTR
and the neo gene is regulated by the SV40 early promoter. MO7e cells
were transduced with control vector, LXSN, or L(ASp21)SN. Cell lines
stably expressing antisense p21 sequence (AS21) or the control vector
(LXSN) were obtained by culture in the presence of G418.
Antisense p21 mRNA expression decreases steady-state p21 protein
levels.
High levels of p21 mRNA expression in AS21 cells are demonstrated by
Northern analysis (Fig 1A).
Neither parental MO7e cells nor control LXSN cells expressed the
antisense p21 message or detectable levels of the endogenous p21 mRNA.
Reduced p21 protein levels (Fig 1B) resulted from antisense p21
expression in AS21 cells during log phase growth.
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| Fig 1.
Antisense p21 mRNA expression and its effect on
p21 protein levels. (A) The top picure is a Northern blot hybridization
of p21 mRNA expression from parental (MO7e), LXSN (vector control), and
AS21(antisense p21) cells visualized with probe specific to human p21.
The lower picture is ethidium bromide staining of the same blot to show
total RNA loading; 18S and 28S rRNA is indicated. (B) Western blot
visualization of p21 protein in lysates from LXSN and AS21 cells probed
with monoclonal antibody to human p21 (equal amounts of protein were
loaded per lane). Data are representative of two experiments.
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Antisense p21 mRNA expression causes deformed nuclear architecture
and polyploidy.
After growth of the transduced cell lines in G418 was stabilized, we
noted the presence of cells with differing sizes. We found that some
AS21 cells were very large and had multiple apparent nuclei
(Fig 2A).
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| Fig 2.
Effect of antisense p21 mRNA expression on nuclear
morphology. (A) Wright-Giemsa stain of a mononuclear LXSN cells (upper
left). The others are examples of multilobular cells and/or
cells with two, three, or more apparent nuclei from AS21 cultures. (B)
Quantitation of different nuclear morphologies in transduced cells.
Data from three separate cultures were pooled. Two hundred cells per
culture were scored. Mean ± SD of each morphology is compared.
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| Fig 3.
Cells with multiple or deformed nuclei have multiple
copies of some chromosomes. Fluorescence in situ hybridization of log
phase cells using probe to centromere no. 7. Blue shows DAPI-stained
DNA and red shows centromere no. 7 signal indicating the number of
copies of chromosome no. 7. The upper three pictures show examples of
LXSN cells that are mononuclear and are diploid, as indicated by two
copies each of chromosome 7. The AS21 picture (bottom) shows an example
of a single giant cell (center) with 4 nuclear lobular structures. Two
of these lobes contain several copies of chromosome 7; therefore, this
cell is polyploid.
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In the AS21 cells, there was a pronounced shift in the percentage of
cells with grossly deformed or multiple nuclei compared with control
LXSN cells (Fig 2B). Because p21 has been linked to megakaryocyte
differentiation,18 we considered whether AS21 cells were
beginning a megakaryocytic differentiation program. Electron
photomicrographs could not confirm separate multiple nuclei, but did
show that many apparent nuclei were connected by a single nuclear
membrane (data not shown). These micrographs also showed what appears
to be structures similar to  granules, but there was no difference
in their frequency between these two cell lines. No evidence of a
demarcation membrane system could be seen, and it therefore appears
that the change in morphology is not due to megakaryocytic
differentiation. This suggestion is supported by flow cytometric
analysis of CD61 and CD41 surface expression using anti-CD61 and
anti-CD41 antibodies, which also showed no difference between the two
cell lines (data not shown).
The ploidy of AS21 cells was checked using fluorescence in situ
hybridization and centromeric probes to chromosome 1 (not shown) and 7 (Fig 3). All mononuclear cells observed contained two copies of each of
these chromosomes indicating that they were diploid. However, some of
the giant multinuclear cells found in the AS21 cultures contained
multiple copies of the same chromosome per nucleus demonstrating that
they were polyploid. This suggests that antisense reduced p21
expression can cause increased polyploidy. This finding is supported by
flow cytometric analysis of polyploid cells in LXSN and AS21 cultures
that demonstrate the increase in incidence of polyploid cells in AS21
cultures (Fig 4).
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| Fig 4.
Effects of antisense p21 mRNA expression on polyploidy.
Cells from log phase cultures of LXSN control cells or AS21 cells were
stained with propidium iodide and subjected to cell cycle analysis
according to Materials and Methods. After gating out dead cells and
doublets, cell events in channel numbers greater than the highest 4N
channel were enumerated as a polyploid event. The percentage polyploid
cells is shown. *Significantly different from control, P < .05.
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Reduced p21 expression causes relaxed MT-dependent checkpoints.
p53 and p21 have both been implicated in checkpoints involving
MTs.9-11 Deficiencies in either of these molecules have
been linked to genetic instability and polyploidy, in part through defects in these checkpoints. We considered whether a defective MT
checkpoint could, in part, be responsible for the polyploidy we
observed in AS21 cells. We therefore tested if MT checkpoints were
intact in AS21 cells by treatment with the MT poisons, nocodazole and
colcemid. These agents disrupt the mitotic spindle and activate the
spindle assembly checkpoint and arrest cells in metaphase that can be
seen microscopically and can easily be quantitated by flow cytometric
cell cycle analysis.
A dose-response experiment (Fig 5) using nocodazole
(NOC) showed that low concentrations were more effective than high NOC concentrations in inducing G2 phase (metaphase) arrest in control LXSN
cells. This biphasic dose-response was not apparent in AS21 cells.
However, NOC induced G2 phase accumulation was greater in AS21
cultures. Similar results were obtained with colcemid (data not shown).
Further experiments with high and low NOC concentrations (Fig 6) showed two salient points. (1) High NOC
concentrations cause significantly more G0/G1 and less G2/M phase
arrest than lower concentrations in control cells. (2) NOC induced more
G2/M phase arrest of AS21 cells than control cells at all
concentrations tested. Together, these data show that the mitotic
spindle assembly checkpoint was activated in both cell lines by NOC.
The newly described MT-dependent arrest checkpoint in
G0/G110 also appears to have been activated in control
cells by high NOC, but is defective in AS21 cells. This leads to
greater G2/M accumulation in AS21 cutlures, especially with high NOC
concentrations.
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| Fig 5.
Dose-response effect of nocodazole on G2/M phase
proportion in transduced cells. Log phase cultures were washed with
phosphate buffered saline and put into fresh medium with GM-CSF plus
the indicated amount of nocodazole and incubated for 24 hours. Cells
were then harvested and cell cycle analysis was performed. This
experiment was repeated twice with similar results.
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| Fig 6.
Effect of nocodazole on cell cycle distribution in
transduced cells. Cells were treated as described in Fig 5 except for
48 hours. G0/G1, S, and G2/M DNA contents are shown. Data are the
averages ± SD from three separate experiments. *Significant
difference from untreated control, P < .05. Low NOC was 0.15 µg/mL and high NOC was 15 µg/mL.
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Because high NOC concentrations seem to suppress G0/G1 exit, we
compared NOC allowable G1 exit in LXSN and AS21 cells as represented by
the percentage of decrease of G0/G1 phase induced by NOC treatment compared with control (Table 1). After 24 hours, NOC
treatment significantly inhibited G1 exit (negative percentage of
decrease). After 48 hours of treatment, G1 exit in LXSN cells occurred
in the presence of low NOC, but was still suppressed by high NOC. On
the other hand, significant G1 exit of AS21 cells occurred even in the
presence of high NOC. This further supports the idea that antisense p21
expression relaxes an MT-dependent cell cycle checkpoint in G1 phase.
A more definitive confirmation of a relaxed G1 MT checkpoint is shown
in
Fig
7. Cells were synchronized in G0/G1 phase by growth-factor deprivation
arrest and then released from this arrest in the presence or absence of
NOC. Cell cycle analysis was then performed 24 hours later, which is
within the first cycle after release. It is seen that high NOC
treatment suppressed G1 exit in control LXSN cells but did not suppress
G1 exit in antisense expressing AS21 cells. We therefore suggest that
antisense p21 expression relaxes an MT polymerization-dependent cell
cycle arresting checkpoint in G1 phase.
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| Fig 7.
Effect of high and low nocodazole treatment on the cell
cycle in synchronized LXSN and AS21 cells. Cells were synchronized by
growth factor starvation for 18 hours and then released by adding back
growth factor (GM-CSF) in the presence or absence of the indicated
amount of nocodazole and incubation for 24 hours before cell cycle
analysis. Nocodazole concentrations were the same as described in Fig
5. The 4N proportion at time zero was negligible. Data are
representative of three separate experiments.
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NOC has been shown previously to induce p21.19 We therefore
investigated if NOC treatment upregulated p21 in our cell lines and if
antisense p21 expression suppressed this induction.
Figure 8 shows the results of Western blot
analysis of p21 protein levels after NOC treatment. p21 was induced by
NOC, and this response was significantly attenuated by antisense p21
expression. This then suggests that reduced p21 protein induction is
responsible for the MT checkpoint relaxing effects of antisense p21
sequence expression.
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| Fig 8.
Effect of nocodazole on p21 protein levels in transduced
cells. (A) Example of a Western blot of whole cell lysate using
anti-p21 antibody. (B) Quantitation and statistical analysis of three
experiments like that in (A). Data are expressed as the percentage
above background density. The mean ± SD is shown. *Statistically
significant difference compared with control LXSN cells (P < .05). NOC concentrations were the same as described in Fig 5.
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The human G0/G1 phase MT checkpoint requires p21.
p21 was previously reported to be required for G0/G1 arrest after
mitotic slippage after spindle damage11 and also for
another MT-dependent G0/G1 arrest,10 both in murine
embryonic fibroblasts. The effects of NOC have not been reported in p21
null human cells. Some murine checkpoints are different than the same
human checkpoints, and because a model human cell line was used, we
wanted to verify the G1 MT checkpoint in human p21/
cells. Figure 9 shows the effects of NOC on
wild-type and p21/ human colorectal cell lines. Low NOC
caused G2/M phase arrest, whereas high NOC treatment resulted in more
G0/G1 and less G2/M arrest in wild-type cells, which is similar to the
dose-response observed in LXSN cells. In contrast, there was a greater
increase in G2/M arrest in p21/ cells compared with
wild-type cells at both NOC concentrations. This was similar to but
more pronounced than the same dosing effect found in AS21 cells. In
addition, there was an increase in the 8N (polyploid) cells in treated
p21/ cultures. This indicates a loss of the re-replication block in the p21 null cells.
Figure 10 is an analysis of G1 phase exit
in one heterozygous and two different clonal p21 null human cell lines
that was performed in a manner similar to that reported in Table 1.
There was no statistically significant difference between the wild-type
and the heterozygous cell line, but there was a significant increase in
NOC allowable G1 exit in both of the p21 null cell lines. Furthermore,
the G1 exit reducing effects of high NOC were observed in wild-type
cells, but not in the knock-out cells, a dose-response similar to that
observed in AS21 cultures. From these data it is concluded that human
p21 null cells have a similar, but more robust, loss of the G1 MT checkpoint as that found in AS21 cells, which is also similar to that
reported for murine p21 null cells. Also, p21 is required for the
prevention of re-replication in response to mitotic slippage in the
presence of NOC in these human cells.
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| Fig 9.
Effect of nocodazole treatment on cell cycle profile in
human colorectal cell lines. Log phase culture of wild-type
(p21+/+) or p21 null (p21/) cells were treated with the
indicated amount of nocodazole for 24 hours and then cell cycle
analysis was performed. 2N(G0/G1), 4N(G2/M), and 8N(polyploid) DNA
contents are shown (data are representative of 3 separate
experiments).
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| Fig 10.
Effect of nocodazole on p21 deficient human colorectal
cell lines. Wild-type, heterozygous (p21+/), and two different
homozygous p21-deficient cell lines were treated as described in Fig 8
and then cell cycle analysis was performed. G0/G1 exit was calculated
as in Table 1. Mean ± SD from three separate experiments are shown.
*Statistically significant difference from wild-type response
(P < .05).
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Antisense p21 mRNA expression causes centriole overduplication.
The centrosome is the MT organizing center in mammalian cells. p53
deficiency results in centrosome amplification and nuclear abnormalities similar to that observed in AS21 cells.20 p53 activates p21 expression, especially during G1 phase, when the centrioles duplicate.21 This line of evidence caused us to
investigate the status of the centrosome in AS21 cells, especially
since this has not been reported in human p21-deficient cells.
Figure 11 shows three
examples of cells with abnormal nuclei after staining with -tubulin.
-Tubulin localizes to the centrioles. Cells in control LXSN cultures
were mononuclear and displayed one or two centriolar/centrosomal signals as exemplified in the two upper cells in the top picture of Fig
11. Almost every cell in AS21 cultures that we identified in this
fashion that had more than one apparent nucleus displayed supernumerary
centriolar signals as exemplified in the top picture and other
pictures. Therefore, it is concluded that, similar to p53 deficiency,
p21 deficiency results in abnormal centriole replication.
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| Fig 11.
Effects of antisense p21 mRNA expression on
centrosome distribution. These three pictures show examples of cells
containing nuclei and stained in situ with antibody to -tubulin,
which localizes the centrosome (red foci). The nuclei are visualized
with DAPI (blue). The insets are phase contrast images of the same
cells stained with Giemsa to show the cellular boundries. The upper
picture shows three cells. The upper two cells are mononuclear cells
and demonstrate a single red centrosomal focus (see text for further
descriptions). The lower cell contains a deformed nucleus and
demonstrates a cluster of several centrosomal foci. This type of cell
was found exclusively in AS21 cultures. The other two pictures show
further examples of cells with deformed nuclei also containing several
centrosomal foci each.
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DISCUSSION |
During our investigations of the effects of hematopoietic growth
factors on the cell cycle and on cell cycle regulating
molecules,12,13 we generated a factor-dependent human cell
line that has suppressed p21 expression. We subsequently noted a
remarkable change in the nuclear morphology of many of these cells.
Anitsense expressing cultures contained cells with more than one
apparent nucleus, and many cells had strangely shaped, multilobed
nuclei. This could not be explained by megakaryocytic differentiation,
at least within the context of surface marker expression. However,
because the parental MO7e cell line already expresses some
megakaryocytic markers, an effect of antisense p21 on megakaryocytic
differentiation remains possible. Increased p21 was previously reported
to be linked with increased megakaryocytic
differentiation.18 Because we reduced p21 expression and
found no change in surface markers, our findings do not necessarily
contradict this report. The megakaryocyte-like changes in nuclear
morphology we observe after antisense p21 mRNA expression could be
solely due to miscoordination of mitotic or other cell cycle
checkpoints. However, this remains to be shown.
p21 has been previously linked to chromosomal positioning and
intranuclear chromatin structure.22 Our data suggest that p21 is also important to gross nuclear architecture. p21 is considered to be important to the maintenance of genetic stability because of its
tumor-suppressor function.23 Our findings of increased polyploidy and deformed nuclei in p21-deficient human cells are consistent with this.
p21 is required for MT surveillance cell cycle checkpoints.
The p53/p21 axis has been shown to be involved in several cell cycle
checkpoints,3,4,8-11 including the mitotic spindle assembly
checkpoint, and a newly described MT damage checkpoint in G1 phase, as
well as a G1 checkpoint that blocks DNA re-replication. As a potential
cause of the polyploidy observed in AS21 cells, these MT checkpoints
were investigated by treatment of cells with nocodazole. Nocodazole
activated the spindle assembly checkpoint in both AS21 and vector
control cells, as noted by the increased arrest of cells in G2/M phase
measured by DNA analysis and as noted by increased metaphase cells
observed microscopically (data not shown). Increasing concentrations of
nocodazole caused less G2/M arrest and more G0/G1 arrest in control
cells, which is consistent with a hightened activation of a G1 MT
checkpoint. AS21 cells showed an apparent loss of the G1 MT
checkpoint-activating effects of high concentrations nocodazole
treatment. A relaxed G1 MT checkpoint in AS21 cells was substantiated
using G0/G1 phase synchronized cells. The requirement of p21 for G1
arrest and re-replication blockage in response to high nocodazole was
further demonstrated with p21-deficient human colorectal cell lines.
Together, these data support the existence of an MT damage checkpoint
in G1 phase in human hematopoietic cells and show that p21 is required
for the G1 phase arresting effects of this checkpoint. Because
suppressed p21 induction in G1 phase in response to MT disruption
results in a defective G1 cell cycle arrest checkpoint, it likely
contributes to the observed polyploidy found in AS21 cells. This
checkpoint therefore contributes to genetic stability during hematopoiesis.
The p53/p21 axis couples centriole replication to DNA replication.
Spatial organization and cell polarity are maintained by the
cytoskeletal proteins, including the MTs.24 The centrosome is the focal point for MT organization.25 p53 deficiency
has been shown to result in centrosome amplification and grossly
deformed nuclear morphologies similar to those observed in AS21
cells.20 However, before now, p21-deficient cells had not
been tested for this effect. Our data show that AS21 cells with
multiple or deformed nuclei have supernumerary centrioles. p21
overexpression has recently been reported to inhibit centriole
replication in amphibian embryonic cells.26 We have now
documented the converse of this by showing that reduced p21 expression
promotes centriole overduplication in human hematopoietic cells. This
strongly suggests that the p53/p21 axis is important to the
coordination of the centriole duplication cycle with the DNA
replication cycle. Loss of this coordination could account for the
deformed nuclei observed in AS21 cells and could therefore contribute
to the generation of aneuploidy frequently observed in leukemia and
other human cancers.
The following points can now be considered together. (1) The p53/p21
axis is important for MT checkpoints. (2) Centrioles organize
interphase MTs and the mitotic spindle. (3) The p53/p21 axis is
important to centriole replication. (4) The centrioles duplicate in G1
phase. (5) MTs are important for centriole replication. When considered
together, this line of evidence raises the intriguing possibility that
the G1 MT checkpoint that depends on the p53/p21 pathway may in fact be
a centriole/centrosome duplication checkpoint.
MTs are the target for one of the most widely used and successful class
of chemotherapy agents (antimitotics). Tumor-specific killing by
chemotherapy agents has been linked to loss of cell cycle
checkpoints.5 A centrosome duplication checkpoint could potentially be targeted by novel chemotherapeutic strategies.
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ACKNOWLEDGMENT |
The authors thank Kent Robertson, MD, and Robert Hromas, MD, for the
retroviral vector; Scott Cooper for help with illustrations; Perluigi
Porcu, MD, for megakaryocyte assays; Yu Tian for Northern blot
analysis; and Cindy Booth for secretarial assistance.
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FOOTNOTES |
Submitted June 8, 1998; accepted October 14, 1998.
Supported by US Public Health Grants No. R01 HL56416, R01 DK53674, and
R01 HL54037 and by a Project in P01 HL53586 from the National
Institutes of Health to H.E.B. S.E.B. is a Fellow of the Leukemia
Society of America, Inc.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Charlie Mantel, Walther
Oncology Center, R4-325, 1044 W Walnut St, Indianapolis, IN 46202-5121;
e-mail: cmantel{at}topaz.iupui.edu.
| |
REFERENCES |
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Hartwell LH, Weinert TA:
Checkpoints: Controls that ensure the order of cell cycle events.
Science
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Abstract
Introduction