1 Center for Evolutionary Genetics CNR, c/o Department of Genetics and Molecular
Biology, University `La Sapienza', Via degli Apuli 4, 00185 Rome, Italy
2 Department of Biology, CB#3280, University of North Carolina at Chapel Hill,
Chapel Hill, NC 27599, USA
* Author for correspondence (e-mail: f.degrassi{at}caspur.it)
Accepted 1 November 2001
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Summary |
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Key words: Lagging chromosomes, Merotelic orientation, Mono-orientation, Mitotic checkpoint, Aneuploidy
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Introduction |
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The integrity of the cell and of its genome and the correct accomplishment
of cellular processes depend on the existence of control points in the cell
cycle. These control mechanisms, called `checkpoints', inhibit the transition
to the next cell cycle phase if the events of the previous phase have not been
correctly executed. A mitotic checkpoint has been identified that controls the
metaphase to anaphase transition. A large number of studies have demonstrated
that in vertebrates the kinetochore plays an active role in the mitotic
checkpoint pathway and that microtubule accumulation at the kinetochore and/or
tension that microtubules produce on the attached kinetochore can be the
signal for mitotic checkpoint inactivation
(Rieder and Salmon, 1998).
Most of the mitotic checkpoint proteins localize on kinetochores during the
early stages of mitosis (prophase and prometaphase) and disappear or become
partially depleted from kinetochores before anaphase onset. These proteins can
also be detected on unattached kinetochores of mono-oriented chromosomes at
late prometaphase (Chen et al.,
1996
; Taylor and McKeon,
1997
; Waters et al.,
1998
; Martinez-Esposito et
al., 1999
). The same behavior has been observed for the
kinetochore phosphoepitopes recognized by the 3F3/2 antibody
(Gorbsky and Ricketts, 1993
;
Campbell and Gorbsky, 1995
).
The mitotic checkpoint inhibits anaphase if spindle assembly is disrupted and
if one or more kinetochores are not attached to spindle fibers. In the
presence of these defects the mitotic checkpoint is activated, preventing
anaphase until all kinetochores become attached
(Rieder and Salmon, 1998
).
Hence, the activation of the mitotic checkpoint prevents the unbalanced
distribution of chromosomes during mitosis and the production of aneuploid
cells.
However, the fidelity of chromosome segregation is not absolute, and
segregation defects, such as chromosomes left near the spindle equator after
anaphase onset (lagging chromosomes) or sister chromatids migrated to the same
pole (nondisjunction), have been observed in cultured mammalian cells
(Ford et al., 1988;
Cimini et al., 1999
;
Catalán et
al., 2000
). A large number of elegant studies in grasshopper
meiosis, dating from more than two decades ago, resulted in a model in which,
although initial kinetochore malorientations are very frequent, they are
efficiently corrected during prometaphase by a process in which kinetochore
microtubules can detach and reattach several times, until the correct
orientation is achieved (Nicklas,
1971
; Bajer and
Molè-Bajer, 1972
;
Nicklas, 1997
). This model
proposes that meiotic or mitotic malsegregation may occur when kinetochore
malorientations are not corrected before anaphase
(Nicklas and Koch, 1969
;
Ault and Rieder, 1992
).
Lagging chromosomes at anaphase represent a potential source of aneuploidy.
After cytokinesis occurs, a lagging chromosome may give rise to a monosomic
daughter cell and a trisomic one in 50% of cases. Several studies have shown
that treatment with low doses of spindle poisons can induce chromosome loss
during mitosis (Hsu and Satya-Prakash,
1985; Rizzoni et al.,
1989
; Marshall et al.,
1996
; Izzo et al.,
1998
; Cimini et al.,
1999
), and defects in chromosome segregation and lagging
chromosomes at anaphase have been observed after inactivation of the mitotic
checkpoint by antibody microinjection (Chan
et al., 1999
; Chan et al.,
2000
). Furthermore, cells subjected to prolonged exposure to high
doses of spindle poisons are not permanently arrested but override the mitotic
checkpoint and undergo a mitotic slippage
(Andreassen and Margolis, 1994
;
Casenghi et al., 1999
),
suggesting that efficiency of the checkpoint may not be absolute. Both the
meiotic studies and the more recent investigations on the mitotic checkpoint
provided the ground for the recurrent idea in the literature that lagging
chromosomes in mitosis may arise in cells that enter anaphase in the presence
of mono-oriented chromosomes (Nicklas,
1971
; Bajer and
Molè-Bajer, 1972
;
Ford, 1985
;
Ault and Rieder, 1992
;
Chan et al., 2000
).
We have recently shown for untreated PtK1 cells and PtK1 cells recovering
from a mitotic block that lagging chromosomes in anaphase are single
chromatids with only one kinetochore, as detected by CREST staining.
Immunofluorescence and electron microscopy studies showed that the single
kinetochore of a lagging chromosome is merotelically oriented (i.e. connected
to microtubule bundles coming from both poles)
(Cimini et al., 2001). In this
work we extend our previous study by investigating whether human primary
fibroblasts exhibit similar segregation errors and analyzing in live imaging
experiments whether progression to anaphase of cells with mono-oriented
chromosomes may contribute to the production of anaphase lagging chromosomes.
In human cells, the use of chromosome-specific alphoid probes in fluorescence
in situ hybridization experiments provides a sensitive tool to follow the
distribution of single sisters from the same chromosome during mitosis and
discriminate loss of a single sister from whole chromosome loss for specific
chromosomes. Fluorescence in situ hybridization with chromosome 7 and 11
alphoid probes showed that single chromatids represent the great majority of
loss events in ana-telophases for human primary fibroblasts released from a
nocodazole-induced mitotic arrest. But loss of both sisters from the same
chromosome is over-represented compared with the expected probability of a
random occurrence of single events. We also analyzed the state of the mitotic
checkpoint phosphoepitope recognized by the 3F3/2 antibody on the kinetochores
of lagging chromosomes in human anaphase cells. We found that they are devoid
of the phosphoepitope, as expected if they attached microtubules and
congressed to the metaphase plate prior to anaphase. Live cell imaging of
mitotic progression in H2B-GFP-transfected human and PtK1 cells provided
direct proof that lagging chromosomes observed in anaphase cells never derive
from a checkpoint over-ride in the presence of mono-oriented chromosomes.
Laggin chromosomes are seen left behind at the spindle equator during anaphase
only, after metaphase chromosome congression, as predicted for lagging
chromosomes having merotelic kinetochore orientation.
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Materials and Methods |
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FISH analysis
MRC-5 cells were treated with 35 ng/ml nocodazole (Sigma) for 16 hours to
arrest cells in mitosis or with 0.125% DMSO, the nocodazole solvent, as
control. At the end of the treatment, cells were washed three times in
pre-warmed medium and then re-incubated in fresh medium at 37°C in order
to release cells from the mitotic block. After 30 or 60 minutes, cultures were
fixed in a 3:1 methanol:acetic acid mixture. FISH staining was performed using
alphoid probes specific for chromosome 7 and 11 as previously described
(Cimini et al., 1997).
Preparations were examined using a Zeiss Axioplan microscope equipped with a
100x (1.3 NA) oil immersion objective and a CCD camera (Photometrics).
DAPI, FITC and Rhodamine fluorescence were detected using the 0.1, 10 and 15
Zeiss filter sets, respectively. Grayscale images were acquired using IP Lab
Spectrum software and processed with Adobe PhotoShop software. On each
coverslip, all ana-telophase cells were visualized, and frequencies and types
of lagging chromosomes were recorded. Frequencies of chromosome loss were
analyzed by pooling data from cultures allowed to recover for 30 or 60 minutes
after nocodazole treatment, since the cumulative frequency of ana-telophases
observed at the two recovery times was found to correspond approximately to
the frequency of mitotically arrested cells observed at the end of nocodazole
treatment (data not shown). Data presented are the sum of two to four
experiments.
Immunostaining with 3F3/2 antibody
MRC-5 cells were treated with nocodazole for 16 hours, released for 30 or
60 minutes and then fixed and immunostained with the 3F3/2 antibody. Cells
were rapidly rinsed in PHEM buffer (60 mM Pipes, 25 mM Hepes, pH 6.9, 10 mM
EGTA, 4 mM MgSO4) and then lysed for 5 minutes in 0.5% Triton-X in
PHEM buffer in the presence of 100 nM microcystin (Calbiochem). Cells were
fixed for 20 minutes in 4% formaldehyde in PHEM, freshly prepared from
paraformaldehyde, then rinsed in PBST (PBS with 0.05% Tween-20) and
subsequently blocked in 10% boiled goat serum in PBS for 1 hour at room
temperature. Coverslips were then incubated 2 hours at 37°C with the 3F3/2
antibody (a generous gift of G. J. Gorbsky, Health Sciences Center, University
of Oklahoma, Oklahoma City, OK), rinsed in PBST and incubated with an FITC
anti-mouse antibody (Vector Laboratories) diluted 1:100 in 5% boiled goat
serum for 1 hour at 37°C. Coverslips were rinsed again, counterstained
with DAPI and mounted in an antifade solution (Vector Laboratories).
Microscope observation and image acquisition were performed as described
above.
Analysis of mitotic progression and chromosome dynamics in live
cells
A plasmid carrying the full-length coding sequence for H2B histone
subcloned into the pEGFP-N1 mammalian expression vector (a generous gift from
Paulo Magalhaes, University of Padua, Italy) was used to express an H2B-GFP
fusion protein under a CMV promoter in MRC-5 and PtK1 cells. MRC-5 cells,
subcultured on 22 mm coverslips in 35 mm Petri dishes, were transiently
transfected with 1 µg plasmid DNA using FuGENE 6 Transfection Reagent
(Boheringer Mannheim). After 30 hours of incubation, cells were rinsed with
PBS to remove the transfection mixture and incubated in 35 ng/ml nocodazole
for 16 hours. Cells were then rinsed three times in medium and observed using
fluorescence and phase-contrast microscopy after 30 minutes post-incubation in
fresh medium. A Nikon Eclipse 300 inverted microscope equipped with a 37°C
heated stage, 60x (0.7 NA) objective, and a Nikon B-2A filter block was
used to follow live cell mitotic progression in MRC-5 cells. Late prometaphase
or metaphase cells were localized and mitotic chromosome dynamics was
observed. Time intervals from anaphase onset to cleavage furrow appearance and
from cleavage furrow appearance to completion of cytokinesis were recorded.
PtK1 cells were transfected as described above and culture medium was
supplemented with 200 µg/ml Geneticin (Gibco BRL) 48 hours after
transfection. Surviving cells at 1 week were maintained under selection using
60 µg/ml antibiotic. After 3 weeks the cell population was highly enriched
(>80%) in cells expressing the H2B-GFP fusion protein. PtK1 cultures
enriched in H2B-GFP transfected cells were arrested in mitosis with 150 ng/ml
nocodazole for 3 hours. Cells were subsequently washed, post-incubated in
drugfree medium for 30 minutes and then observed by fluorescence and
phase-contrast microscopy. A Nikon TE300 inverted microscope equipped with a
37°C heated stage, 100x (1.4 NA) Plan Apo phase 3 objective, a
Chroma Hy-Q FITC filter set, and an Orca 1 CCD camera (Hamamatsu Photonics)
was used to follow mitotic progression in living PtK1 cells by time-lapse
microscopy. The microscope was controlled by MetaMorph imaging software
(Universal Imaging Corp.).
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Results |
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Finally, a statistical analysis of the data showed that observed
frequencies of loss of separated sisters from the same chromosome at anaphase
were at least one order of magnitude higher than expected values for loss of
single sisters from the same chromosome as independent events (chromosome 7
double loss expected value:
(7.37x10-3)x(7.37x10-3)=
54.32x10-6=0.054%; chromosome 7 experimental
frequency: 0.63%
; chromosome 11 expected value:
(7.68x10-3)x
(7.68x10-3)=58.98x10-6=0.059%
;
chromosome 11 experimental frequency: 1.72%
), suggesting that the
cellular mechanism responsible for chromosome loss during mitosis biases the
random loss of sister chromatids.
3F3/2 antibody does not stain kinetochores of anaphase lagging
chromosomes
To investigate the state of an important mitotic checkpoint marker on
anaphase lagging chromosomes in human primary fibroblasts, we immunostained
MRC-5 cells with the 3F3/2 antibody, which recognizes a kinetochore
phosphoepitope involved in the mitotic checkpoint signaling
(Campbell and Gorbsky, 1995).
Mono-oriented chromosomes possess this phosphoepitope on their kinetochores;
it subsequently disappears as chromosomes bi-orient and properly align on the
metaphase plate (Gorbsky and Ricketts,
1993
; Nicklas et al.,
1995
). When tension is applied to a single unaligned chromosome by
micromanipulation, 3F3/2 staining is lost and anaphase begins
(Li and Nicklas, 1995
;
Li and Nicklas, 1997
;
Nicklas, 1997
). Furthermore,
when 3F3/2 antibody is injected into metaphase cells, anaphase onset is
delayed (Campbell and Gorbsky,
1995
).
MRC-5 cells were arrested in mitosis by nocodazole treatment and then fixed
and immunostained after recovery in drug-free medium in order to characterize
the 3F3/2 behavior during mitosis in this human primary cell line and analyze
3F3/2 staining on lagging chromosomes at anaphase
(Fig. 2). As already shown in
other non-human and human cancer cell lines
(Rieder and Salmon, 1998),
unattached kinetochores from nocodazole-blocked MRC-5 cells were highly
enriched in the 3F3/2 phosphoepitope (Fig.
2A), whereas no staining was observed on aligned metaphase
kinetochores (Fig. 2B). 3F3/2
immunodetection on human primary fibroblasts in anaphase showed that
kinetochores of both segregated chromosomes and lagging chromosomes (346 cells
analyzed) did not stain for the 3F3/2 antibody
(Fig. 2C,D), as recently
observed in PtK1 anaphase cells (Cimini et
al., 2001
). This suggests that kinetochores of lagging chromosomes
observed in anaphase cells interacted with kinetochore microtubules at some
point during prometaphase and/or metaphase, becoming dephosphorylated, as
expected for kinetochores of bi-oriented chromosomes, which are under tension,
and for merotelically oriented kinetochores. However, this analysis cannot
exclude the possibility that during the release from the nocodazole-induced
mitotic arrest a fraction of cells over-rides the mitotic checkpoint in the
presence of unattached or mono-oriented chromosomes and the phosphorylated
epitope recognized by the 3F3/2 antibody is dephosphorylated after anaphase
onset.
|
Lagging chromosomes appear during anaphase
To test the possibility that cells showing lagging chromosomes at anaphase
were cells progressing from metaphase to anaphase in the presence of
mono-oriented chromosomes, we performed in vivo experiments to follow mitotic
progression and chromosome dynamics in live cells after nocodazole exposure.
In these experiments we considered chromosome alignment to the metaphase plate
as representative of chromosome biorientation, since live observations in
other cell lines have shown that mono-oriented chromosomes are not aligned at
the spindle equator but are closer to spindle poles; they then congress to the
metaphase plate as they become bi-oriented
(Rieder et al., 1994;
Campbell and Gorbsky, 1995
;
Howell et al., 2000
). The
progression from metaphase to the completion of cytokinesis was followed in
human primary fibroblasts recovering from a nocodazole-induced mitotic arrest,
recording the time spent from anaphase onset to the appearance of the cleavage
furrow (anaphase) and the time spent to complete cytokinesis after cleavage
furrow appearance (telophase/cytokinesis). To follow chromosome dynamics
during mitosis, we transiently transfected human primary fibroblasts with a
vector carrying an H2B-GFP fusion gene
(Kanda et al., 1998
).
Transfected or untransfected human primary fibroblasts were
nocodazole-arrested in mitosis and released for 30 minutes in fresh medium to
allow spindle reassembly; late prometaphase or metaphase cells were then
localized and observed until completion of cytokinesis by H2B-GFP fluorescence
(Fig. 3A) and phase-contrast
microscopy (Fig. 3B). The same
type of experiment was performed on a PtK1 cell population highly enriched in
transfected cells by non-clonal antibiotic selection after transfection
(Fig. 4A (H2B-GFP
fluorescence); Fig. 4B (phase
contrast)). The use of a GFP-tagged histone construct in our work provided a
powerful means to follow chromosome dynamics during mitosis. This was
particularly effective in visualizing the mitotic chromosome behavior in the
MRC-5 human fibroblast cell line, since the small size of human chromosomes,
the three-dimensional shape of the mitotic cells, and the low contrast between
chromosomes and cytoplasm makes the observation of chromosome dynamics by
phasecontrast very ambiguous (Fig.
3B). The analysis of duration of mitosis in untransfected and
H2B-GFP transfected MRC-5 cells showed that the expression of the H2B-GFP
fusion protein does not affect the length of anaphase or telophase in cells
recovering from the nocodazole-induced mitotic block
(Table 1). The analysis of
mitotic progression in H2B-GFP expressing MRC-5 and PtK1 cells provided
important clues for understanding the dynamics of events leading to lagging
chromosomes at anaphase and the fate of these malsegregated chromosomes.
First, none of the cells that showed one or a few unaligned chromosomes
entered anaphase in MRC-5 cells (5 out of 5 cells), or in PtK1 cells (7 out of
7 cells) within the period of live observation (2-3 hours). Second, all
lagging chromosomes that could be observed at anaphase appeared in cells in
which all the chromosomes correctly aligned at the metaphase plate before
anaphase onset (MRC-5 cells: see 60 and 20 minutes in
Fig. 3; PtK1 cells: see 15 and
0 minutes in Fig. 4). Third,
none of the lagging chromosomes we observed was incorporated in the daughter
nuclei during nuclear envelope reformation but instead formed micronuclei
(Fig. 5). This suggests that
the potential aneuploidy caused by lagging chromosomes cannot be rescued by
successful later migration or by passive incorporation of lagging chromosomes
into the main nucleus at the end of mitosis. As a whole, the live observation
of mitotic progression of cells with lagging chromosomes demonstrated that in
both cell lines the mitotic checkpoint is fully proficient in blocking the
metaphase to anaphase transition in cells with mono-oriented chromosomes.
However, it does not prevent the loss of genetic information represented by
merotelically oriented lagging chromosomes.
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The comparison of anaphase and telophase length did not show statistically significant differences between MRC-5 cells with and without lagging chromosomes at anaphase, although a tendency towards longer time in telophase/cytokinesis for cells with lagging chromosomes was observed (Table 1). In PtK1 cells the presence of lagging chromosomes did not affect anaphase length but significantly delayed the completion of cytokinesis (Table 1), suggesting that cytokinesis rather than chromosome migration is affected by the presence of lagging chromosomes in this cell line. It should be noted that the numbers reported as telophase/cytokinesis times in our live observations refer to the minutes elapsed between cleavage furrow formation and the completion of cell cleavage and therefore the measurements reflect more completion of cytokinesis than chromosome migration, which is usually completed before the cleavage furrow forms. In a number of cases we observed chromosomes migrating behind the others that successively reached the segregated chromosomes at one pole, but in these cells anaphase and telophase/cytokinesis length was unaffected (data not shown).
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Discussion |
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In the present study the in vivo observation of mitotic progression during
recovery from nocodazole demonstrates that lagging chromosomes in anaphase are
observed in cells where all the chromosomes correctly aligned at the metaphase
plate. This was found in both PtK1 and human cells, providing a general
significance to the conclusion that chromosome loss in cultured tissue cells
is not due to an over-riding of the mitotic checkpoint in the presence of
mono-oriented kinetochores. Our live cell imaging shows that the mitotic
checkpoint efficiently inhibits anaphase onset in cells with unattached
kinetochores both in human and PtK1 cell lines, thereby preventing the
possible aneuploid burden caused by mono-oriented chromosomes. However, this
control mechanism does not detect kinetochore merotelic orientation, since
this unusual interaction between kinetochores and kinetochore microtubules
inactivates the checkpoint, as shown in this and previous work
(Cimini et al., 2001). Earlier
live cell experiments had already shown merotelic orientation of chromosome
fragments possessing one kinetochore lagging behind at anaphase
(Khodjakov et al., 1997
) or of
chromosomes stranded at the spindle equator in anaphase cells with multipolar
spindles (Sluder et al.,
1997
).
A crucial question for understanding the origin of mitotic instability is
whether the presence of a lagging chromosome in anaphase may activate a kind
of control mechanism, allowing time for correction of this segregation error.
In fission yeast, slowing of spindle elongation in live cells with lagging
chromosomes was recently observed by time-lapse microscopy
(Pidoux et al., 2000).
However, no clear indication for a possible `anaphase checkpoint' was provided
in that paper, which also showed, consistently with our 3F3/2 data, that the
mitotic checkpoint protein Bub1 was not present on lagging chromosomes at
anaphase (Pidoux et al.,
2000
). Our data suggest that an active mechanism that delays
anaphase/telophase in cells with lagging chromosomes is not likely, since no
significant lengthening of the time between anaphase onset and cleavage furrow
formation is observed in human fibroblasts or PtK1 cells that show lagging
chromosomes at anaphase. By contrast, a significant lengthening of the
cleavage process was observed in PtK1 cells. This might be related to the
large size of chromosomes in PtK1 cells, which renders this cell line a
paradigmatic experimental model for studying mitosis and chromosome
segregation. In PtK1 cells the presence of a lagging chromosome may influence
the duration of mitosis by exerting a mechanical impediment to cleavage furrow
ingression and cytokinesis completion, whereas in human cells this may depend
very much on the size of the lost chromosome. Thus, we propose cell cleavage
delay rather than activation of an `anaphase/telophase checkpoint', as the
cause of telophase/cytokinesis lengthening in PtK1 cells with lagging
chromosomes. This is also consistent with the observation that cytokinesis
completion is longer in PtK1 cells with more than one lagging chromosome
compared with cells with a single lagging chromosome (single lagging:
20.29±16.22 minutes; multiple lagging: 27.8±25.9 minutes).
It has been recognized for many years that micronuclei result as the
consequence of the exclusion from daughter nuclei of acentric fragments or
whole chromosomes lagging at anaphase
(Evans, 1988;
Ford et al., 1988
;
Heddle et al., 1991
). The
quantitative relationship between frequencies of acentric fragments and
micronuclei is not precisely known, although it is suggested that between 30
and 70% of the acentric fragments seen in anaphase become micronuclei
(Evans, 1988
;
Heddle et al., 1991
).
Frequencies of the same order of magnitude between whole chromosome lagging at
anaphase and micronuclei have been observed in human lymphocytes, suggesting
that a large part of lagging chromosomes give rise to centromere positive
micronuclei (Ford et al.,
1988
; Gustavino et al.,
1994
). Our in vivo analysis is consistent with the quantitative
relationship between lagging chromosomes and micronuclei inferred from
analyses on fixed preparations. This is a relevant aspect since it means that
the presence of a lagging chromosome at anaphase may lead, in half of cases,
to the formation of a monosomic daughter cell and a trisomic one possessing a
disomic nucleus and an extra chromosome in a micronucleus. It has also been
shown that some micronuclei can perform DNA synthesis and mitotic condensation
synchronously with the main nucleus, further producing chromosome loss and
gain at successive mitotic cycles (Rizzoni
et al., 1989
; Gustavino et
al., 1994
; Minissi et al.,
1999
). This may explain the generation of chromosome instability
after chromosome loss. Accordingly, the presence of micronuclei in interphase
appears to be a common feature of tumor cells
(Saunders et al., 2000
;
Sen, 2000
).
In conclusion, our results demonstrate that the mitotic checkpoint
efficiently prevents the possible aneuploid burden caused by mono-oriented
chromosomes in human primary fibroblast cells and that merotelic kinetochore
orientation is a major limitation for accurate chromosome segregation and a
potentially important mechanism of aneuploidy in human cells. The
identification of cellular mechanisms involved in chromosome malsegregation is
of great interest because of the potential role that aneuploidy has been
suggested to have in tumor development and progression
(Li et al., 1997;
Duesberg et al., 1998
;
Cahill et al., 1998
;
Lengauer et al., 1998
;
Lee et al., 1999
). It
represents an extraordinarily important clue to understand the genesis of
chromosome instability in tumor cells and a starting point for the development
of new therapeutic strategies.
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Acknowledgments |
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