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Brief report
The interphase microtubule damage checkpoint defines an S-phase
commitment point and does not require p21waf-1
Charlie R. Mantel, Stephen E. Braun, Younghee Lee, Young-June Kim, and Hal E. Broxmeyer
Cell cycle checkpoints ensure orderly progression of events during cell division. A
microtubule damage (MTD)-induced checkpoint has been described in G1 phase of
the cell cycle (G1MTC) for which little is
known. The present study shows that the
G1MTC is intact in activated T lymphocytes from mice with the p21waf-1 gene
deleted. However, p21waf-1 gene deletion
does affect the ratio of cells that arrest at
the G1MTC and the spindle checkpoint
after MTD. The G1MTC arrests T lymphocytes in G1 prior to cdc2 up-regulation
and prior to G1 arrest by p21waf-1. Once
cells have progressed past the G1MTC,
they are committed to chromosome replication and metaphase progression, even
with extreme MTD. The G1MTC is also
present in a human myeloid cell line deficient in p21waf-1 gene expression. The
p21-independent G1MTC may be important in cellular responses to MTD such as
those induced by drugs used to treat
cancer. (Blood. 2001;97:1505-1507)
© 2001 by The American Society of Hematology
Introduction
All proliferating animal cells must accurately duplicate and transmit the total of their genome during each cell division. This
remarkable fidelity of chromosome replication and segregation
depends on cell cycle checkpoints,1 especially in highly proliferative cells. Loss of mitotic checkpoint function has been linked to
the origin and progression of human malignancy.2 The microtubule/
mitotic spindle assembly checkpoint (SAC) is essential to normal
growth and development in mice.3 Signaling molecules of the SAC
have garnered intense recent interest because mutations in these
mitotic checkpoint genes have been demonstrated in human
cancers.2 Moreover, the tumor suppressor, p53, which is mutated in
more than half of all human tumors, along with one of its
downstream effectors, p21waf-1 (p21), are required for proper cell
cycle arrest after spindle disruption.4-7
We recently described a human growth-factor dependent hematopoietic cell line that is defective in levels of p21 expression
(AS21), especially in response to microtubule damage (MTD).7
These cells display a similar, albeit less penetrant, defect in mitotic
checkpoints compared with human cells completely lacking p21. In
those studies, p21 was implicated in loss of both G1 and M phase
MTD checkpoints, observations further substantiated in human
colorectal cell lines with p21 gene deletion. We proposed that p21
was important for proper SAC responses and involved in a new
interphase MTD checkpoint (G1MTC).
We have now investigated by intracellular flow analysis the
requirement for p21 in the G1MTC using proliferating murine T
lymphocytes with the p21 gene deleted and AS21 cells. We find
that p21 gene is not required for the G1MTC arrest, but it is
involved in regulating G1 progression past the G1MTC commitment point. This point is now defined as the point at which cdc2 is
up-regulated in G1 phase and the cell becomes refractory to
G1 arrest by MTD and becomes committed to completion of
From the Departments of Microbiology/Immunology, Walther Oncology Center,
Indiana University School of Medicine, and the Walther Cancer Institute,
Indianapolis, IN.
Submitted July 28, 2000; accepted October 4, 2000.
Supported by Public Health Service grants RO1 HL 56416 and RO1 DK 53674
to H.E.B.
BLOOD, 1 MARCH 2001 䡠 VOLUME 97, NUMBER 5
DNA replication and mitotic initiation, even in the presence of
extreme MTD.
Study design
Cells, antibodies, and treatments
The MO7e cells containing antisense p21 (AS21) or empty vector (LXSN)
were generated and maintained as described.7,8 Asynchronously growing
cells were treated with 15 ␮g/mL nocodazole or taxol (Sigma Chemical, St
Louis, MO), or with control diluent for 24 hours, then analyzed by
multivariate flow cytometric cell cycle analysis. For flow cytometry,
fluorescein-isothiocyanate (FITC)-conjugated antihuman/mouse cdc2 rabbit polyclonal IgG1 or rabbit FITC-IgG1 isotype control antibodies were
used to determine specific and nonspecific cdc2 immunofluorescence
intensity, respectively. Activated splenic T lymphocytes were obtained9
from p21⫺/⫺ and p21⫹/⫹ mice.10,11
Multivariate flow cytometric cell cycle analysis
Flow cytometric analysis was performed on cells treated with nocodazole,
taxol, or diluent by first fixation and simultaneous permeabilization using
Cytofix/Cytoperm reagent (Pharmingen, San Diego, CA) according to the
manufacturer’s instructions. After washing to remove the fixative, intracellular staining was done by incubating the cells for 1 hour with 10 ␮g/mL
FITC-conjugated antibody to cdc2 or with 10 ␮g/mL FITC-conjugated
isotype control antibody. After washing 3 times, the intracellularly labeled
cells were counterstained with propidium iodide (Sigma) for 30 minutes to
quantitate DNA. Cellular fluorescence intensity was measured with a
FACscan flow cytometer (Becton-Dickinson, San Jose, CA). Laser light
scatter was used to gate out dead cells. Cell cycle proportions were
calculated using the Modfit computer program (Verity Software House,
Topsham, ME). Density scatter diagrams were constructed and analyzed
using the WinMDI program (J. Trotter, The Scripps Research Institute, La
Jolla, CA, http://facs.scripps.edu/). Other details including multicolor
Reprints: Charlie R. Mantel, Walther Oncology Center, 1044 West Walnut St,
Indianapolis, IN 46202-5121; e-mail: [email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2001 by The American Society of Hematology
1505
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1506
MANTEL et al
BLOOD, 1 MARCH 2001 䡠 VOLUME 97, NUMBER 5
compensation have been described.12 Statistical comparisons used the
Student t test. Experiments were performed at least 3 times.
Results and discussion
Figure 1 shows cdc2 expression in activated murine T lymphocytes
as a function of DNA content. The cdc2 content is up-regulated in
G1 phase. There are at least 2 separate populations of G1 cells with
respect to cdc2 levels, G1a and G1b. Cells enter S and G2/M phases
with very little further increase in cdc2 expression. After mitosis,
cdc2 is degraded and the daughter cells return to G1 phase with
little or no cdc2. A 3-fold shift in the relative proportions of G1a and
G1b cells is observed in the cells with the p21 gene deleted. This is
consistent with the proposed role of p21 in G1 phase progression
and “threshold” events as reported.11,13 After treatment with the MT
depolymerizing agent, nocodazole, the G1b population could not be
observed in wild-type or p21 knockout mouse cells (Figure 1C,D).
However, the relative proportion of G2/M phase cells was higher in
the p21 knockout cells after treatment compared to wild-type cells.
There was also an increase in the number of 8N cells in the p21
knockout cultures after nocodazole treatment compared to wildtype cells.4-7 Treatment with taxol resulted in a similar response
(not shown). We interpret this as indicating that G1a cells are
arresting after MTD at a point in G1 phase before cdc2 expression is
up-regulated. Once cells have progressed in the cell cycle past this
point, and cdc2 expression is turned on, subsequent MTD no longer
arrests the cells in G1, but they progress through S and G2 phases to
arrest at the SAC in mitosis. Thus, the point of cdc2 up-regulation
defines the point of passage of the G1MTC when cells become
committed to DNA replication, even in the presence of MTD. The
absence of p21 does not appear to influence G1 arrest by the
G1MTC. However, the temporal effect of p21 deletion on the
proportions of cells at G1a versus G1b leads to fewer cells that arrest
at the G1MTC after MTD, and leads to the increased proportion of
p21 knockout cells arrested in mitosis as reported.4-7
An identical experiment was performed on p21-deficient human
myeloid AS21 cells,7 to determine if the human G1MTC arrest is
also independent of p21 (Figure 2). A similar pattern of cdc2
expression occurred during cell cycle progression in these cells as
observed in murine T lymphocytes. The relative proportions of G1a
and G1b cells are shifted just as in the mouse cells (Figure 2A,B)
and after treatment with nocodazole or taxol (not shown), the G1b
population disappears with a commensurate shift in the G1 and
G2/M arrested proportions (Figure 2C,D).
An interesting finding is the appearance of a population of 4N
Figure 1. Expression of cdc2 and cell cycle arrest of T lymphocytes after MTD.
Scatter density diagrams of bivariate (cdc2/DNA) cell cycle analysis of activated T
lymphocytes from wild-type mice (p21⫹/⫹; A,C) and p21 knockout mice (p21⫺/⫺; B,D)
are shown. After activation, cells were treated for 24 hours with 15 ␮g/mL nocodazole
(C,D) or control diluent (A,B). 2N (G0/G1) and 4N (G2/M) DNA content is indicated
along with the percentages of cells in different cell cycle phases. Data are
representative of at least 2 replicate experiments from 6 sets of mutant mice and their
wild-type littermate controls.
Figure 3. Two MTD-induced cell cycle checkpoints. This illustration shows the
proposed relative location of the 2 known cell cycle arrest points in cells with MTD.
SAC indicates spindle assembly checkpoint; G1MTC, G1 phase microtubule checkpoint. The centrosome cycle (1 or 2 MTOC indicates microtubule organizing centers)
is shown to be coordinated with the DNA/chromosome cycle (2N or 4N DNA content).
The arrests the cell cycle in G1 phase between the shift from a low cdc2-expressing
state to a high cdc2-expressing state. Some of the proteins implicated in each MT
checkpoint are listed (see reference 15 for a recent review).
Figure 2. Expression of cdc2 and cell cycle arrest of human myeloid cell line,
MO7e, after MTD. Scatter density diagrams of bivariate cell cycle analysis of vector
control cells (LXSN; A,C) and p21 antisense (AS21; B,D) are shown. Day 3 cell
cultures were treated for 24 hours with 15 ␮g/mL nocodazole (C,D) or control diluent
(A,B). The 2N (G0/G1) and 4N (G2/M) DNA content is indicated along with the
percentages of cells in different cell cycle phases. Data are representative of at least
6 separate experiments done in triplicate.
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BLOOD, 1 MARCH 2001 䡠 VOLUME 97, NUMBER 5
THE INTERPHASE MICROTUBULE CHECKPOINT
human cells with low (Figure 2C) or negative (Figure 2D) cdc2
content that is not observed in murine cells. This population could
represent p21-deficient cells that have prematurely exited mitosis
without cytokinesis and thus have escaped cell cycle arrest induced
by SAC activation, suggesting a difference between human and
murine responses to MTD. Embryonic cells from p21⫺/⫺ mice have
been reported to have an intact SAC response, but do fail to prevent
re-replication of DNA4 events consistent with our analysis. On the
other hand, loss of p53 in a human tumor cell line is reported to be
without effect on nocodazole arrest.6 Rodent cells are known to
have “leaky” or missing cell cycle checkpoints compared to human
cells. Also, somatic cells may have different or additional checkpoints compared to embryonic cells. Therefore, cell type and
species differences in response to MTD are possible. These issues
are currently under investigation.
Questions yet to be answered are: What is the purpose of the
G1MTC and what is the nature of the MTD sensing mechanism?
The sensing apparatus of the SAC is believed to reside at the
kinetochore, and tension on 2 juxtaposed MT attachment points is
believed to be the key mechanochemical process that senses correct
1507
chromosome-spindle alignment and sends a “go” signal for anaphase initiation and chromosome congression.14 Because the
centrosome is the MT organizing center of interphase cells, and
because this organelle must duplicate and separate in G1 phase
ultimately to become the poles of the mitotic spindle, we speculate
that the G1MTC defines a cell cycle checkpoint ensuring proper
centriole duplication and separation. Figure 3 illustrates the
“location” of the G1MTC in relation to the SAC, the centrosome
cycle, and the DNA cycle. The relationship between the G1MTC
and the restriction point, or other cell cycle commitment points or
checkpoints, remains to be determined. However, the existence of
an interphase MTD checkpoint has significant implications in
treatment strategies of cancer by drugs that exert selective toxicity
on cancer cells by interfering with MT dynamics.
Acknowledgment
The authors wish to thank Patricia Mantel for help in editing the
manuscript.
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2001 97: 1505-1507
doi:10.1182/blood.V97.5.1505
The interphase microtubule damage checkpoint defines an S-phase
commitment point and does not require p21 waf-1
Charlie R. Mantel, Stephen E. Braun, Younghee Lee, Young-June Kim and Hal E. Broxmeyer
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