Mutagenesis vol.14 no.5 pp.513–520, 1999
Induction of polyploidy and apoptosis after exposure to high
concentrations of the spindle poison nocodazole
Berlinda Verdoodt, Ilse Decordier, Karen Geleyns,
Mónica Cunha, Enrico Cundari and
Micheline Kirsch-Volders1
Laboratory for Cell Genetics, Vrije Universiteit Brussel, Pleinlaan 2, 1050
Brussels, Belgium
The proportions of aneuploid/polyploid versus euploid cells
formed after treatment with spindle poisons like nocodazole
are of course dependent on the relative survival of cells
with numerical chromosome aberrations. This work aimed
at studying the survival of polyploid cells formed after
treatment with a nocodazole concentration sufficient to
significantly decrease tubulin polymerization (0.1 µg/ml).
First, normal primary lymphocytes were analysed and the
following complementary chromosomal parameters were
quantified: mitotic index, frequency of abnormal mitoses,
polyploid metaphases and apoptotic cells. The results clearly indicate a positive correlation between abnormal
mitotic figures, apoptosis and the induction of polyploidy.
They therefore led to a single cell approach in which both
apoptosis and polyploidy induction could be scored in the
same cell. For this purpose, actively proliferating cells are
required and two human leukaemic cell lines were used,
KS (p53-positive) and K562 (p53-negative), which have
a near-triploid karyotype. Cells were separated into an
apoptotic and a viable fraction by means of annexinV staining and flow cytometry. In KS, treatment with
nocodazole induced a similar fraction of hexaploid cells in
both the viable and apoptotic fraction, but no dodecaploid
cells were ever observed. In contrast, a population of
dodecaploid cells (essentially viable) was clearly observed
in the K562 cell line. The results in KS, as compared with
K562, confirm that wild-type p53 can prevent further
cycling of polyploid cells by blocking rereplication. The
most probable explanation for these data is that not only
the mitotic spindle but also interphase microtubules are
sensitive to nocodazole treatment. Our data thus strongly
suggest that besides the G1/S checkpoint under the control
of p53, the G2/M transition may be sensitive to depolymerization of microtubules, possibly under the control of Cdc2,
Bcl-2, Raf-1 and/or Rho.
Introduction
Polyploidy, which corresponds to a change to an exact multiple
of the haploid number of chromosomes (e.g. 4n and 8n), can
be induced via two main routes: rereplication of the DNA in
the absence of an intervening mitosis or in the absence of a
functional spindle or premature exit from mitosis to the next
G1 phase without having completed chromatid migration to
the poles (for a review see Kirsch-Volders et al., 1998). The
consequences of polyploidy may be directly dependent on the
ploidy status, as for abnormal sperm cells or oocytes, which
1To
are responsible for spontaneous abortions, increased transcription rate in particular tissues (liver and trophoblast) or predisposition to oncogenic transformation (Pathak et al., 1994).
Since polyploidization in tumours is often followed by aneuploidy, which is in turn associated with high grade invasive
tumours and poor prognosis (Sandberg, 1977; Giaretti, 1994;
Segers et al., 1994; Verdoodt et al., 1994), the mechanisms
leading to polyploidy and the survival of polyploid cells have
recently been attracting increasing interest.
Polyploidy can be induced by certain chemicals; these
compounds can often, but not always, also induce aneuploidy
(for a review see Aardema et al., 1998). The methodologies
to assess polyploidy are similar to those available to detect
aneuploidy (for a review see Kirsch-Volders et al., 1998). Both
events can therefore be studied in parallel. An example of
chemicals that can induce both aneuploidy and polyploidy are
the so-called spindle poisons, compounds that interfere with
the formation of the metaphase spindle. Nocodazole, which
influences microtubule turnover (Jordan et al., 1992; Vasquez
et al., 1997) and which has been used for chemotherapy,
belongs to this group of products. In isolated human lymphocytes analysed with the in vitro cytokinesis blocked micronucleus test, Elhajouji et al. (1995, 1997, 1998) demonstrated
that nocodazole did induce aneuploidy and at higher doses
also polyploidy.
The proportions of aneuploid/polyploid versus euploid cells
observed are of course also dependent on the relative survival
of cells with numerical chromosome aberrations. Until now,
induction of programmed cell death specifically in these
aneuploid/polyploid cells during the following interphase and
in the next mitosis was not our object of study. Since our
earlier data (Cundari et al., 1996; Cundari et al., submitted
for publication) suggested that aneuploid cells induced by
lower nocodazole concentrations survive relatively well, in
this work we studied the survival of polyploid cells formed
after treatment with a higher nocodazole concentration (0.1
µg/ml). Our aim was also to try to identify the signals
responsible for apoptosis after nocodazole treatment and to
determine the phases of the cell cycle in which it is induced.
In a first approach, normal primary human lymphocytes
were studied to avoid any bias due to the aneuploid character
of transformed permanent cell lines. Complementary chromosomal parameters were quantified: frequency of abnormal
mitotic figures and apoptotic cells during the first and second
mitotic wave and frequencies of polyploid metaphases at the
second mitosis. The results clearly indicate a positive correlation between abnormal mitotic figures, apoptosis and polyploidy.
This positive correlation between apoptosis and abnormal
metaphases or polyploidy in lymphocytes led to a single cell
approach in which both events can be scored in the same cell.
For this purpose, actively proliferating cells are required and
two human leukaemic cell lines were used, KS and K562.
whom correspondence should be addressed. Tel: 132 2 629 34 23; Fax: 132 2 629 27 59; Email: [email protected]
© UK Environmental Mutagen Society/Oxford University Press 1999
513
B.Verdoodt et al.
K562 is often used as a model cell line for the study of
apoptosis (see for example Kaufmann et al., 1993; McGahon
et al., 1994; Durrieu et al., 1998; Esteve et al., 1998). KS was
derived from K562 by means of infection with H1 parvovirus.
This virus preferentially kills tumoral cells; cells surviving
infection with H1 have a suppressed malignancy. KS was
found to re-express wild-type p53 (Telerman et al., 1993); the
gene is present in K562, but is not expressed. These specific
cell lines were selected as there are indications that p53 has a
role in the prevention of aneuploidy and polyploidy.
Cells in which a functional spindle cannot be formed
normally arrest in metaphase (Rieder et al., 1994; Sluder et al.,
1994). This arrest is, however, only temporary and cells
eventually exit metaphase to become aneuploid or polyploid
(reviewed in Rudner and Murray, 1996). It has already been
found that p53-negative cells become aneuploid or polyploid
at a higher frequency when treated with spindle poisons (Cross
et al., 1995; Minn et al., 1996; Di Leonardo et al., 1997),
suggesting a role for p53 in the regulation of polyploid cell
propagation via activation of a post-mitotic checkpoint (Lanni
and Jacks, 1998).
In both cell lines, the following chromosomal parameters
were analysed: frequencies of polyploid metaphases in the
presence of nocodazole and frequencies of polyploidy versus
aneuploidy in apoptotic and viable interphase cells during up
to three cell cycles of nocodazole treatment. The results
confirm that, in the presence of nocodazole, p53 can block the
rereplication of DNA in polyploid cells. Moreover, it was
demonstrated that polyploid and euploid cells are present
in similar proportions in viable and apoptotic cells before
rereplication. This suggests that apoptosis is triggered before
mitotic segregation has taken place. The possibility that the
absence of polymerized tubulins at the G2/M transition can
also induce apoptosis, possibly under the control of Cdc2, Bcl2, Raf-1 and/or Rho, is also discussed.
Materials and methods
Cell culture
Human peripheral blood samples were obtained from healthy donors of ,30
years of age. Lymphocytes were isolated using Ficoll-Pacque (Pharmacia
Biotech, Uppsala, Sweden) and were cultured at a concentration of 0.53106
cells/ml in Ham’s F-10 medium with 25 mM HEPES buffer and l-glutamine
(Gibco BRL, Paisley, UK), supplemented with 15% fetal calf serum (Gibco
BRL), 1% penicillin/streptomycin (Gibco BRL) and 2% phytohaemagglutinin
HA16 (PHA; Murex Biotech Ltd, Dartford, UK). For the chromosome number
counting, colcemid (GIibco BRL) was added at 0.2 µg/ml culture 1.5 h before
terminating the cultures.
The human chronic myelogenous leukaemia cell line K562 (Lozzio and
Lozzio, 1975) and its subclone KS (Telerman et al., 1993) were grown in
RPMI 1640 medium (Life Technologies, Paisley, UK) supplemented with
10% heat-inactivated fetal calf serum (Gibco BRL) in an atmosphere with
5% CO2 and 100% humidity at 37°C. These cell lines are near-triploid in
untreated cultures (Lozzio and Lozzio, 1975).
Nocodazole, dissolved in dimethylsulfoxide (DMSO), was added to the
lymphocyte cultures 24 h after the start of culture. The final DMSO
concentration did not exceed 0.5%. Primary lymphocyte cultures were harvested after a total culture time of 48 or 72 h, corresponding to 24 and 48 h
of nocodazole exposure; exponentially growing cultures of K562 and KS were
treated for 24, 48 and 72 h. Only 0.5% DMSO was added to control cultures.
Cells were fixed with 3:1 methanol/acetic acid. Slides were prepared by
dropping the fixed cell suspension onto them; they were stored at –20°C until
use. For chromosome counting and classification of mitotic figures, slides
were stained in 5% Giemsa solution (Merck) in Sörensen buffer, pH 6.8
(Prosan, Gent, Belgium). Scattered chromatin as defined in Kirsch-Volders
(1986) actually corresponds to apoptotic nuclei. Cells were counted at a
magnification of 12503 on a Zeiss transmitted light microscope.
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TUNEL assay
The In Situ Cell Death Detection kit AP (Boehringer-Mannheim, Mannheim,
Germany) was used according to the instructions of the manufacturer, both
for the primary lymphocytes and for both cell lines. Slides were counterstained
with ethidium bromide, to be visualized with epifluorescence microscopy at
a magnification of 4003.
Annexin staining and flow sorting
Cells were collected by centrifugation and resuspended in 100 µl annexin
labelling solution, consisting of 2% annexin-V-FLUOS (BoehringerMannheim) and 0.1 µg/ml propidium iodide in HEPES buffer (10 mM HEPES/
NaOH, 140 mM NaCl, 5 mM CaCl2, pH 7.4). Cells were incubated in the
dark in this solution for 15 min. When used for counting aneuploid cells, they
were spread onto slides and air dried, but not otherwise fixed. If cells were
going to be used for flow sorting, they were resuspended in HEPES buffer
and no propidium iodide was added to the labelling solution. Cells were
separated into apoptotic (annexin-positive) and viable (annexin-negative)
populations, using a FACstar PLUS flow cytometer (Becton Dickinson,
Oxford, UK).
In situ hybridization
The pUC1.77 probe (Cooke and Hindley, 1979), which hybridizes to the
satellite III pericentromeric region of chromosome 1, was used to detect
chromosome 1. To label the centromere of chromosome 17 the probe D17Z1
was used; this probe was obtained from ATCC (Rockville, MD).
Probes were labelled by nick-translation with digoxigenin-11-dUTP (Boehringer-Mannheim) or biotin-11-dUTP (Boehringer-Mannheim). The labelling
reaction took place in a total volume of 50 µl for 1 µg of probe with 1.5
nmol of dATP, dCTP and dGTP and 3 nmol of digoxigenin-11-dUTP or the
same amount of biotin-11-dUTP, adding DNase I/DNA polymerase I. Products
were acquired in the form of a kit (Nick Translation System; Gibco BRL).
The reaction was carried out at a temperature of 15°C for 2 h.
Fluorescence in situ hybridization (FISH) was essentially carried out as
described by Viegas-Pequignot et al. (1989). Briefly, slides were treated with
0.01% RNase in 23 SSC (0.3 M sodium chloride, 0.03 M sodium citrate) for
60 min at 37°C, rinsed in 23 SSC, then treated with 50 µg/ml pepsin in 10
mM HCl for 10 min at 37°C and finally dehydrated in a graded ethanol series
(50, 70 and 98%) and air dried. Aliquots of 40 ng of both probes, dissolved
in 20 µl of a hybridization mix containing 50% formamide, were used per
slide; slides and probe were denatured together for 4 min at 90°C on a
hotplate. Hybridization took place overnight at 37°C.
After hybridization, slides were washed twice for 7 min in 23 SSC, 50%
formamide and twice for 2 min in 23 SSC, both at 43°C, after which they
were rinsed in blocking buffer (0.5% blocking reagent in 43 SSC; BoehringerMannheim). The slides were then incubated first with avidin–FITC (Vector
Laboratories), at 1/200 dilution, then with monoclonal mouse anti-digoxigenin
(Boehringer-Mannheim) at a dilution of 1/250 together with anti-avidin (Vector
Laboratories) at a dilution of 1/100, and finally again with avidin–FITC, at
1/200 dilution with Texas Red®-conjugated sheep anti-mouse antibody (Amersham, Little Chalfont, UK) at a dilution of 1/20. All incubations took place
at 37°C for 30 min. In between incubations, slides were washed in 43 SSC,
0.05% Tween 20. After dehydration in a graded alcohol series, the nuclei
were counterstained with DAPI at a concentration of 1 µg/ml.
To score the FISH slides, the criteria as defined by Hopman et al. (1990)
were applied: spots were counted on non-overlapping intact nuclei and
secondary spots, which give a much weaker signal, were ignored. Cells with
more than nine spots for either chromosome were counted as one category,
as it was difficult to distinguish acurately between such high numbers of spots
within one cell.
Statistics
For statistical analysis of the counts of apoptotic cells and abnormal and
polyploid metaphases Fisher’s exact test was used. For the comparisons of
the numbers of FISH signals in the cell lines either Fisher’s exact test or the
χ2 test were used, depending on the number of categories. The significance
of correlation coefficients is determined by a test based on the normal
distribution.
Results
Positive correlations between abnormal mitotic figures, polyploidy and apoptosis after in vitro nocodazole treatment in
human primary lymphocytes
Induction of abnormal mitotic figures
Treatment of PHA-stimulated lymphocytes for 24 or 48 h with
nocodazole at concentrations of 0.04 and 1.0 µg/ml induced a
Nocodazole, polyploidy and apoptosis
Fig. 1. Changes in the fraction of normal and abnormal mitotic figures after nocodazole treatment for 24 and 48 h in primary human lymphocytes. , normal
mitoses; , C mitoses; , anaphase lagging and dislocated chromosomes; , chromatin masses. The columns indicate the mean value of both cultures, the
lines the values for the two separate cultures.
Table I. Mitotic figures counted on the total number of cells in nocodazoletreated primary lymphocytes
Treatment
Mitotic index
Normal mitoses
(% of total no.
of cells)
C mitoses
(% of total no.
of cells)
24
24
24
48
48
48
12.45
22.65
22.02
6.90
28.00
36.10
7.67
13.50
12.05
4.73
10.30
13.00
0.47
3.90
4.71
0.55
9.25
11.40
h,
h,
h,
h,
h,
h,
control
0.04 µg/ml
0.1 µg/ml
control
0.04 µg/ml
0.1 µg/ml
statistically significant increase in the fraction of abnormal
metaphases and anaphases, as compared with the total number
of mitotic cells (Figure 1). In each case, two parallel cultures
from the same donor were counted, which gave similar results,
although the difference between the two cultures was always
larger at the highest nocodazole dose. Per culture, 1000–1800
cells and 68–384 mitoses were counted. The increase in the
fraction of abnormal metaphases and anaphases was time
dependent; the differences between the 24 and 48 h treatments
were highly significant for both the treatment with 0.04 (P ,
0.0001) and with 0.1 µg/ml nocodazole (P , 0.0001). Differences in effect between the two nocodazole concentrations
were less clear and only significant for the shortest treatment
time (P 5 0.0022). The high fraction of chromatin masses in
the control cultures after 24 h treatment is surprising. These
might represent (early) apoptotic cells or be an indication of
toxicity. More advanced techniques would be required to
determine their exact origin.
The increased mitotic index (Table I) with the longer
treatment time was mainly due to an increase in the fraction
of C mitoses. More surprising is that the number of normallooking mitoses also increased.
Induction of apoptosis
On the same slides that were used for the counting of normal
and abnormal mitotic figures, apoptotic cells were also scored
as scattered chromatin. A clear time-dependent increase in the
frequency of apoptotic cells was found by scoring the Giemsa
stained slides (Figure 2). No pronounced differences were
observed between the two nocodazole concentrations studied
(0.04 and 0.1 µg/ml), although the differences were statistically
significant (P , 0.0001 for both treatment times). However,
the treatment duration had a much stronger effect on the
frequency of apoptotic cells. Even in the controls, more
apoptosis was observed after 72 than after 48 h in culture.
The spontaneous induction of apoptosis in cultures of longer
duration may be due to the fact that this is a more or less
artificial system, as compared with the human body, their
normal environment. The correlation between the frequencies
of abnormal metaphases/anaphases and those of apoptotic
nuclei on the same Giemsa stained slides was quite high (r2 5
0.665, P . 0.05), but not significant.
Similar effects were seen when TUNEL staining was used
to detect apoptotic cells on separate slides prepared from the
same donor (Figure 2). Here, 1500–2000 cells were scored
per slide. Assessments of apoptosis with Giemsa or TUNEL
staining correlated very well (r2 5 0.963, P , 0.01), although
the fraction of apoptotic cells detected by TUNEL staining
tended to be higher in the controls and with the shorter
treatment. The differences between the frequency of apoptosis
detected by TUNEL and Giemsa staining may be due to
detection of different stages of aneuploidy; TUNEL is likely to
detect earlier stages which are not yet grossly morphologically
abnormal. For the TUNEL staining, the correlation with the
fraction of abnormal mitoses was somewhat weaker (r2 5
0.608, P . 0.05) than with Giemsa staining.
Polyploidy induction in colcemid-blocked human lymphocytes
In a separate experiment, the frequencies of polyploid metaphases were analysed after in vitro treatment of human lymphocytes with nocodazole (Figure 3). Chromosome counting was
performed on metaphases blocked with colcemid 48 or 72 h
after stimulation with PHA and continuous treatment with
nocodazole (0.04 and 0.1 µg/ml) for 24 and 48 h, respectively.
The data from this experiment clearly show that no polyploidy
was observed in untreated primary lymphocytes: at 24 h
treatment, of 130 metaphases from two parallel cultures, none
was found to be polyploid; at 48 h treatment, no polyploid
metaphases were seen either. The percentage of polyploid
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Fig. 2. Frequency of apoptotic cells in cultures of normal lymphocytes treated with nocodazole, as detected after Giemsa staining ( ) and by TUNEL staining
( ). The columns indicate the mean value of both cultures, the lines the values for the two separate cultures.
Fig. 3. Frequencies of polyploid metaphases in nocodazole-treated metaphases; comparison between 24 and 48 h treatment. The columns indicate the mean
value of the two cultures, the lines the values for the separate cultures.
metaphases obtained after nocodazole treatment was higher
after 48 (3.5 and 8%, respectively) than after 24 h (0.5 and
5%, respectively) treatment. The difference in frequency of
polyploid metaphases between the two treatment times was
significant for 0.04 µg/ml nocodazole only (P 5 0.0335). The
fractions of polyploid metaphases do not appear very high,
but it must be taken into account that there was a strong
accumulation of cells in metaphase in these cultures, pointing
to a delay in the cell cycle. Moreover, stimulation with PHA
does not induce all cultured lymphocytes to divide. A high interculture variability was observed at the higher concentration, for
both treatment times. Except for one octoploid cell, all polyploid cells were tetraploid.
Genetic identity of apoptotic and non-apoptotic cells in
human cell lines expressing or not expressing p53
Polyploidy does not always trigger apoptosis, but loss of
p53 allows further divisions of polyploid human leukaemic
cells treated with nocodazole
To identify the genetic content of apoptotic versus viable cells,
the copy numbers of chromosomes 1 and 17 were determined
by FISH in both the KS and the K562 cell lines after 24, 48
and 72 h treatment with 0.1 µg/ml nocodazole. Before applying
FISH, the cells were separated into apoptotic and viable cells
by means of flow sorting after annexin staining. Annexin
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staining is based on a reversal of cell membrane asymmetry
and detects early apoptotic cells. This is important, as in later
stages of apoptosis we observed that the DNA is too degraded
to obtain meaningful FISH signals.
As a control, cells of both cell lines that had been cultured
without nocodazole for all studied treatment durations were
also subjected to FISH for chromosome 1. The variation in
the distribution of hybridization signals between cultures was
considerable and appeared somewhat larger in the p53-negative
cell line K562 than in the p53-positive KS cells. Nevertheless,
the results show that the major cell population is trisomic for
chromosome 1 in both cell lines (58–89% of cells) but that
tetra- and pentasomic cells are not rare (5.44–21.20 and 2.30–
8.90% of cells, respectively). The distribution of the number
of spots per nucleus did not change in any clear direction with
culture time, but varied rather a lot between cultures, indicating
karyotypic instability in these cell lines. There were no
indications of a spontaneous increase in ploidy. These data
confirm the results of Lozzio and Lozzio (1975), who also
found these cell lines to be near-triploid. A doubling of the
chromosome number thus leads to hexaploid cells. An extra
control culture was not added to the experiments where cells
were separated by FACS, as the fraction of apoptotic cells was
always too low to give meaningful results (unpublished results).
Nocodazole, polyploidy and apoptosis
Fig. 4. Fluorescence in situ hybridization for chromosomes 1 and 17 in viable ( ) and apoptotic ( ) cells of the KS and K562 cell lines. Cells were treated
with 0.1 µg/ml nocodazole for 24, 48 or 72 h. The x-axis indicates the number of spots for both chromosomes; different, different number of spots for the
two chromosomes (aneuploid cells) in the same nucleus.
Interestingly, the proportion of cells treated with nocodazole
with different numbers of spots for chromosome 1 versus
chromosome 17 was very low and never exceeded 15% (Figure
4). This type of signal indicates aneuploid rather than polyploid
cells. Moreover, the difference in the number of signals for
both chromosomes was never greater than two. It was also
independent of the treatment duration or of whether the cells
were viable or apoptotic. These data indicate that at the applied
concentration, nocodazole essentially induces polyploidy.
In KS, treatment with nocodazole shows an induction of
hexaploid cells at the three time points considered, but no
dodecaploid cells were observed after any of the treatments
(Figure 4). This was the case for both viable and apoptotic
cells. The ratio of hexaploid to triploid cells did not change
much over time in the viable cells. In the apoptotic cells, the
fraction of hexaploid cells was clearly higher after 48 (P ,
0.0001) and 72 h (P 5 0.0006) than after only 24 h treatment.
Here the cells with four and five spots have been included in
the triploid population, as such cells also occur in non-treated
cultures of these cell lines.
In contrast, a population of dodecaploid cells (essentially
viable) was clearly observed at 48 and 72 h in the K562 cell
line (Figure 4). A small fraction of dodecaploid apoptotic cells
was only found at 72 h. The fraction of hexaploid cells was
highest after 48 h treatment, but then decreased, concomitant
with the increase in the fraction of dodecaploid cells; the
fraction of triploid cells decreased to a lower level than in KS
at 48 and 72 h treatment. These data show that polyploid cells
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B.Verdoodt et al.
Fig. 5. Frequencies of polyploid metaphases in nocodazole-treated metaphases; comparison between 24, 48 and 72 h treatment in the two cell lines:
K562; , KS. The bars indicate the mean value of the two cultures.
did not preferentially accumulate in the apoptotic subpopulation, as would be expected if polyploidy constituted a strong
signal for the induction of apoptosis.
Survival of polyploid metaphases in the human p53 nonexpressing leukaemia cell line K562 after in vitro treatment
with nocodazole for 72 h
Chromosome counting performed on metaphases blocked with
colcemid 48 or 72 h after stimulation with PHA and continuous
treatment with nocodazole (0.04 and 0.1 µg/ml) clearly showed
(Figure 5) that polyploidy is present in non-treated cells of
these cell lines, between 2 and 9% in K562 and between 6
and 15% in KS. Polyploidy in these cell lines means a multiple
of the triploid chromosome complement, as these cells are
near-triploid in control conditions. In the treated cells the
percentage of polyploid metaphases is dependent on the cell
line, as well as on the treatment. In K562, the p53 nonexpressing cell line, the maximum frequency of polyploid
metaphases was very high (40%) after the longest nocodazole
treatment; polyploidy induction is time but not concentration
dependent. In KS, the p53-expressing cell line, the highest
frequency of polyploid metaphases was 23%; here also induction depends on treatment duration but not on nocodazole
concentration. For these experiments, 100 metaphases were
counted per slide.
Discussion
The major reasons to consider that aneuploidy and polyploidy
are compatible with normal cell life and do not always induce
apoptosis are found in the survival of aneuploid individuals
and polyploid tissues, like the liver (Sateer et al., 1988) in
humans. On the other hand, the idea that the induction of
aneuploidy or polyploidy could trigger apoptosis emerged from
different types of considerations and observations: (i) in a
given tissue the maintenance of a stable karyotype is the goal
of mitotic division and spontaneous segregation errors should
be eliminated; (ii) in mammalian cell cultures in vitro apoptotic
cells were observed after treatment with nocodazole (Wahl
et al., 1996; Cundari et al., submitted for publication); (iii)
the contradictory results described by the ECETOC working
party on chemically induced aneuploidy in which, after treatment with spindle inhibitors, almost no polyploidy was
observed after the first mitotic cycle in primary cells but quite
518
,
clearly occurs in permanent cell lines (Aardema et al., 1998);
(iv) our own quantitative data on the respective frequencies of
chromosome non-disjunction, chromosome loss and polyploidy
induced in vitro in human lymphocytes by spindle inhibitors
(Elhajouji et al. 1995, 1997, 1998).
Given these earlier observations (Elhajouji et al. 1995, 1997,
1998), our aim was to define which genomic changes induced
by nocodazole treatment lead to apoptosis, and in which
phase(s) of the cell cycle. With this objective in mind, we first
studied the correlations between the induction of polyploidy,
apoptosis and abnormal divisions in normal primary human
lymphocytes, to avoid any bias due to the aneuploid character
of most transformed cell lines. We found a positive correlation
between the frequencies of abnormal mitotic figures and of
apoptotic nuclei. Whether these abnormal divisions were all
lethal or whether they might in some cases lead to polyploid
cells was investigated by classical chromosome counting in
metaphase. Our results indicate that polyploid cells are indeed
formed and survive, since a low number of polyploid metaphases was already seen after 48 h in culture and a higher
percentage was found at 72 h. Moreover, an increase in the
mitotic index was also observed in the presence of nocodazole,
which suggests that no strong selective pressure was operating
against mitotic cells.
The positive correlation observed between apoptosis, abnormal metaphases and polyploidy in human primary lymphocytes
led us to the choice of a system in which apoptosis and
polyploidy can be assessed in the same cell. We decided to
do a sorting of apoptotic versus non-apoptotic cells. However,
this methodology requires actively proliferating cells in relatively large amounts. We therefore selected two cell lines of
leukaemic origin: K562 (Lozzio and Lozzio, 1975) and its
derivative KS (Telerman et al., 1993). KS expresses the wildtype p53 gene, whereas K562 does not. K562 has been used
before by different laboratories as a model for the study of
the role of p53 in apoptosis (see for example Kaufmann et al.,
1993; McGahon et al., 1994; Durrieu et al., 1998; Esteve
et al., 1998).
As the induction of polyploid metaphases in these cell lines
did not seem to depend on the nocodazole concentration,
only the higher concentration (0.1 µg/ml) was used for the
comparison of apoptotic and viable interphase cells. In KS,
the cell line which expresses wild-type p53, at 24 h most
Nocodazole, polyploidy and apoptosis
Fig. 6. Some of the genes involved in the induction of apoptosis after
treatment with spindle poisons.
viable cells already showed six spots for both chromosomes
1 and 17, indicating that a first round of polyploidization had
taken place. At both later time points a higher fraction of
trisomic viable cells was seen than at 24 h; no cells with more
than nine spots for either chromosome were ever observed in
this cell line. Of the apoptotic cells, a relatively high fraction
was still trisomic, although the major part was hexasomic.
This would indicate that these cells do not progress to higher
levels of polyploidy than one doubling of the chromosome
number.
The K562 cell line, on the other hand, does not express
p53. In this cell line, few trisomic cells undergo apoptosis at
24 h; the great majority of apoptotic cells were hexasomic,
which remains the case for all further time points. The viable
cells in this line evolved from mainly hexasomic cells at 24 h
to an increasing fraction of dodecasomic cells at 48 and 72 h.
This would indicate that, although some cells die after a first
attempted division with a damaged spindle, other cells are
apparently able to undergo a second division cycle in these
circumstances and to survive further.
After 24 h nocodazole treatment, only a weak increase in
the frequency of polyploid metaphases could be seen and only
at the highest concentration in both cell lines. This is not
surprising, as these cells are not synchronized and have a cell
cycle time of ~19 h. Only a few cells will have had time to
reach a second metaphase in the 24 h period, especially as a
delay in mitosis is to be expected after treatment with a spindle
poison. The fraction of polyploid metaphases clearly increased
with treatment time, as increasing numbers of cells had had
time to go through more than one metaphase. The frequency
of polyploid metaphases did not seem to depend on the
nocodazole concentration in either cell line, however. From
these data it would seem that both concentrations are about as
effective in disturbing the function of the metaphase spindle,
although neither is able to completely inhibit the polymerization
of tubulin (Jordan et al., 1992).
To correctly interpret the data on the frequency of polyploidy
in the apoptotic versus the non-apoptotic cells, it is important
to realise that the cells which were analysed after 48 h
treatment derive from the non-apoptotic cells observed after
24 h treatment; the same applies for the cells analysed after
72 h treatment, which derive from the surviving cells studied
after 48 h treatment. Our results show that after 24 h treatment
polyploid cells either survive or undergo apoptosis, independent
of the presence of p53 protein. This suggests that p53 is not
the only source of an apoptotic signal. Interestingly, the
distribution of spots in apoptotic cells at each time point
mimics that of the viable cells at the preceding time. In a
second phase, the cells which survived after 24 h treatment
will again either survive longer or undergo apoptosis. In the
absence of the p53 gene product these cells can continue to
cycle, as is indicated by the appearance of dodecasomic cells.
This clearly confirms that wild-type p53 can block further
cycling of polyploid cells by blocking rereplication. The
distribution of FISH signals after 72 h treatment confirms the
results observed after 48 h treatment. If the observation that
p53 can block rereplication of the DNA in polyploid cells is
not new (Minn et al., 1996), these results are the first to
demonstrate that the fraction of polyploid cells is almost the
same in apoptotic as in non-apoptotic cells during the hours
which precede the first rereplication cycle.
The most probable explanation for induction of apoptosis
by nocodazole before the passage of polyploid cells through
rereplication is that not only spindle microtubules but also
interphase microtubules are sensitive to nocodazole treatment.
Sensitivity to this spindle poison appears to be greatest at
initiation of mitosis, when the microtubules progress from a
stable, less dynamic interphase organization to the more
dynamic organization of the mitotic spindle (Lieuvin et al.,
1994). Our data thus strongly suggest that besides the G1/S
checkpoint under the control of p53 (for a review see Wahl
et al., 1997), the G2/M transition may be sensitive to depolymerization of microtubules. This seems to correspond to earlier
data (Donaldson et al., 1994; Haldar et al., 1997) on the
effects of taxol on the induction of apoptosis, where G2/M
cells were most sensitive to the effects of this chemical.
Apoptosis induced by spindle poisons is likely independent of
p53 function: KS and K562 underwent apoptosis with about
equal frequencies after nocodazole treatment (Cundari et al.,
submitted for publication).
Other genes have been found to be involved in the apoptotic
pathway after spindle poison treatment (Figure 6); these include
Cdc2 and some of its regulators, genes of the Bcl-2 family,
the c-erbB-2 oncogene, Raf-1 and Rho (Donaldson et al.,
1994; Minn et al., 1996; Blagosklonny et al., 1997; Huang
et al., 1997; Esteve et al., 1998; Yu et al., 1998). An important
factor appears to be the inappropriate activation of Cdc2, i.e.
when the cell is not ready to enter mitosis. This activation can
be prevented (possibly indirectly) through up-regulation of
p21Waf1; c-erbB-2 is able to produce this effect (Yu et al.,
1998). On the other hand, modulation of the effects of genes
of the Bcl-2 family in response to spindle poison treatment
has also been observed. Raf-1, a kinase that is activated by
Ras, can in this case phosphorylate Bcl-2. This phosphorylation
inactivates the anti-apoptotic action of Bcl-2 (Blagosklonny
et al., 1997). Another effector in this pathway is Rho, a Rasrelated GTPase. It has been found to promote apoptosis in
response to serum starvation; this effect is counteracted by
Bcl-2. Finally, up-regulation of the anti-apoptotic Bcl-2 family
member Bcl-xL can prevent the induction of apoptosis after
nocodazole treatment (Minn et al., 1996).
Apart from providing a better insight into the mechanisms
of apoptosis, these results may also aid the development of
antitumour agents, as two of the most successful anti-mitotic
519
B.Verdoodt et al.
agents used in chemotherapy (the vinca alkaloids and the
taxanes) exert their effect through suppression of the dynamics
of the microtubules.
Acknowledgements
The authors wish to thank Prof. P. Caillet-Fauquet and Dr M. Tuynder, of the
Radiobiology Laboratory of the Université Libre de Bruxelles, for the generous
gift of the KS and K562 cell lines. This work was supported by EU research
programme ENV4-CT97-0471.
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Received on March 8, 1999; accepted on May 18, 1999