From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
p21 cip-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
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 antisenseexpressing 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.
r 1999 by The American Society of Hematology.
H
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 factordependent cell line MO7e was synergistically stimulated to
proliferate with the combination of steel factor and granulocytemacrophage 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.
EMATOPOIESIS 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
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.
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.
Address reprint requests to Charlie Mantel, Walther Oncology
Center, R4-325, 1044 W Walnut St, Indianapolis, IN 46202-5121;
e-mail: [email protected].
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.
r 1999 by The American Society of Hematology.
0006-4971/99/9304-0026$3.00/0
1390
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 GMCSF 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 1/1) cells and one clonal heterozygous
(p211/2) deficient and two different clonal homozygous (p212/2)
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
Blood, Vol 93, No 4 (February 15), 1999: pp 1390-1398
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
p21 AND MICROTUBULE DAMAGE CHECKPOINTS
A
B
1391
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-g 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.
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
;
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.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1392
MANTEL ET AL
with differing sizes. We found that some AS21 cells were very
large and had multiple apparent nuclei (Fig 2A).
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 a 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).
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 6 SD of each morphology is compared.
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.
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
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.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
p21 AND MICROTUBULE DAMAGE CHECKPOINTS
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 F .05.
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).
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
1393
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.
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
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.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1394
MANTEL ET AL
Table 1. G1 Exit in the Presence of Nocodazole
G1 Exit
LXSN
% G1
24 Hours
Untreated
Low NOC
High NOC
48 Hours
Untreated
Low NOC
High NOC
65 6 5
64 6 8
76 6 6
70 6 4
44 6 7
65 6 11
AS21
% Decrease
% G1
% Decrease
2
1.5
217.0*
61 6 3
54 6 6
60 6 7
2
11.5
1.6
2
37.1*
7.1
56 6 4
14 6 2
26 6 3
2
75.0*
53.6*
G1 exit was calculated from cell cycle analysis data by determining
the percentage decrease of the G0/G1 proportion of nocodazoletreated cultures compared with untreated control cultures. This reflects the proportion of cells that could progress from G0/G1 phase
and enter S and G2/M phases and was used as a measure of the G0/G1
phase arresting effects of nocodazole. Data are the mean 6 SD from
three experiments.
*Statistically different from untreated control (LXSN), P , .05.
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.
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
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 6 SD from three separate experiments. *Significant difference
from untreated control, P F .05. Low NOC was 0.15 mg/mL and high
NOC was 15 mg/mL.
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.
NOC has been shown previously to induce p21.19 We
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.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
p21 AND MICROTUBULE DAMAGE CHECKPOINTS
1395
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 p212/2 cells. Figure 9 shows the effects of NOC on
wild-type and p212/2 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
p212/2 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 p212/2
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
A
B
Fig 9. Effect of nocodazole treatment on cell cycle profile in
human colorectal cell lines. Log phase culture of wild-type (p211/1)
or p21 null (p212/2) 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).
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 6 SD is shown. *Statistically significant difference compared with control LXSN cells (P F .05). NOC
concentrations were the same as described in Fig 5.
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.
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
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1396
MANTEL ET AL
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.
Fig 10. Effect of nocodazole on p21 deficient human colorectal cell
lines. Wild-type, heterozygous (p211/2), 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 6 SD from three separate experiments are shown.
*Statistically significant difference from wild-type response (P F .05).
with abnormal nuclei after staining with g-tubulin. g-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. 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 g-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.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
p21 AND MICROTUBULE DAMAGE CHECKPOINTS
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 p21deficient 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.
1397
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). Tumorspecific 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.
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.
REFERENCES
1. Hartwell LH, Weinert TA: Checkpoints: Controls that ensure the
order of cell cycle events. Science 246:629, 1989
2. Murray AW: Creative blocks: Cell cycle checkpoints and feedback
controls. Nature 359:599, 1992
3. Livingstone LR, White A, Sprouse J, Livanos E, Jacks T, Tristy
TD: Altered cell cycle arrest and gene amplification potential accompany loss of wild-type p53. Cell 70:923, 1992
4. Kuerbitz SJ, Plunkett BS, Walsh WV, Kastan MB: Wild type p53
is a cell cycle checkpoint determinant following irradiation. Proc Natl
Acad Sci USA 89:7491, 1992
5. Waldman T, Zhang Y, Dillehay JY, Kinzler K, Vogelstein B,
Williams J: Cell cycle arrest versus cell death in cancer therapy. Nat
Med 3:1034, 1997
6. El-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R,
Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B: WAF1, a
potential mediator of p53 tumor suppression. Cell 75:817, 1993
7. Harper JW, Adami GR, Wei N, Keyomarski K, Elledge SJ: The
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1398
p21 cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclindependent kinases. Cell 75:805, 1993
8. Elledge SJ: Cell cycle checkpoints: Preventing an identity crisis.
Science 274:1664, 1996
9. Cross SM, Sanchez CA, Morgan CA, Schimke MK, Ramel S,
Idzerda RL, Raskind WH, Reid BJ: A p53-dependent mouse spindle
checkpoint. Science 267:1353, 1995
10. Khan SH, Wahl GM: p53 and Rb prevent re-replication in
response to microtubule inhibitors by mediating a reversible G1 arrest.
Cancer Res 58:396, 1998
11. Lanni JS, Jacks T: Characterization of the p53-dependent post
mitotic checkpoint following spindle disruption. Mol Cell Biol 18:1055,
1998
12. Mantel CR, Luo Z, Canfield J, Braun S, Deng C, Broxmeyer HE:
Involvement of p21 and p27 in the molecular mechanisms of steel
factor-induced proliferative synergy in-vitro and of p21 in the maintenance of stem/progenitor cells in-vivo. Blood 88:3710, 1996
13. Braun SE, Mantel C, Rosenthal M, Cooper S, Liu L, Robertson
KA, Hromas R, Broxmeyer HE: A positive effect of p21cip-1/waf-1 in the
colony formation from murine myeloid progenitor cells as assessed by
retroviral-mediated gene transfer. Blood Cells Mol Dis 24:137, 1998.
14. Hendrie PC, Miyazawa K, Yang YC, Langefeld CD, Broxmeyer
HE: Mast cell growth factor (c-kit ligand) enhances cytokine stimulation of proliferation of human factor dependent cell line, MO7e. Exp
Hematol 19:1031, 1991
15. Waldman T, Kenzler KW, Vogelstein B: p21 is necessary for the
p53-mediated G1 arrest in human cancer cells. Cancer Res 55:5187,
1995
16. Miller AD, Roseman GJ: Improved retroviral vectors for gene
transfer and expression. Biotechniques 7:980, 1989
MANTEL ET AL
17. Miller AD, Buttimore C: Redesign of retrovirus packaging cell
lines to avoid recombination leading to helper virus production. Mol
Cell Biol 6:2895, 1986
18. Kikuchi J, Furukawa Y, Iwase S, Terui Y, Nakamura M,
Kitagawa S, Kitagawa M, Komatsu N, Miura Y: Polyploidization and
functional maturation are two distinct processes during megakaryocytic
differentiation: Involvement of cyclin-dependent kinase inhibitor p21 in
polyploidization. Blood 89:3980, 1997
19. Tishler RB, Lamppu DM, Price BD: Microtubule-active drugs
taxol, vinblastine, and nocodazole increase the levels of transcriptionally active p53. Cancer Res 55:6021, 1995
20. Fukasawa K, Choi T, Kuriyama R, Rulong S, Vande Woude GF:
Abnormal centrosome amplification in the absence of p53. Science
271:1744, 1996
21. Robbins E, Jentzsch G, Micali A: The centriole cycle in
synchronized HELA cells. J Cell Biol 36:329, 1968
22. Linares-Cruz G, Bruzzoni-Giovanelli H, Alvaro A, Roperch JP,
Tuynder M, Schoevaert D, Nemani M, Prieur S, Lethrosne F, Piouffre L,
Reclar V, Faille A, Chassoux D, Dausset J, Amson RB, Calvo F,
Telerman A: p21waf1 reorganizes the nucleus in tumor suppression. Proc
Natl Acad Sci USA 95:1131, 1998
23. Chen YQ, Cipriano SC, Arenkiel JM, Miller FR: Tumor suppression by p21waf1. Cancer Res 55:4536, 1995
24. Schliwa M: The Cytoskeleton. New York, NY, Springer-Verlag,
1986
25. Mitchison T, Kirschner M: Microtubule assembly nucleated by
isolated centrosomes. Nature 312:232, 1984
26. Stearns T, Winey M: The cell center at 100. Cell 91:303, 1997
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1999 93: 1390-1398
p21cip-1/waf-1 Deficiency Causes Deformed Nuclear Architecture, Centriole
Overduplication, Polyploidy, and Relaxed Microtubule Damage
Checkpoints in Human Hematopoietic Cells
Charlie Mantel, Stephen E. Braun, Suzanna Reid, Octavian Henegariu, Lisa Liu, Giao Hangoc and Hal E.
Broxmeyer
Updated information and services can be found at:
http://www.bloodjournal.org/content/93/4/1390.full.html
Articles on similar topics can be found in the following Blood collections
Neoplasia (4182 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of
Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.