Mutagenesis vol.17 no.2 pp.111–117, 2002
Nature of anaphase laggards and micronuclei in female
cytokinesis-blocked lymphocytes
Ghita C.-M.Falck1, Julia Catalán1,2 and Hannu Norppa1,3
1Laboratory
of Molecular and Cellular Toxicology, Department of Industrial
Hygiene and Toxicology, Finnish Institute of Occupational Health,
Topeliuksenkatu 41 a A, FIN-00250 Helsinki, Finland and 2Department of
Anatomy, Embryology and Genetics, University of Zaragoza, Zaragoza,
Spain
We used pancentromeric fluorescence in situ hybridization
and X chromosome painting to characterize late anaphase
aberrations in cultured (72 h) female lymphocytes in the
presence of cytochalasin B (Cyt-B). Aberrant cells, mostly
containing laggards, were very common (34.5%) among
multipolar anaphases but fewer (5.4%) among bipolar
anaphases. Characterization of the laggards showed that
75% were autosomes, 15% autosomal fragments and 10%
X chromosomes in bipolar divisions; similar figures were
obtained in multipolar cells. The X chromosome lagged
behind more often than would be expected by chance
(1/23), representing 12 and 7% of all lagging chromosomes
in bipolar and multipolar divisions, respectively. Bipolar
divisions contained more lagging autosomes but fewer
lagging fragments and X chromosomes with Cyt-B than
without it. Comparison of the frequencies of anaphase
laggards and interphase micronuclei (MN) showed that
lagging autosomes seldom form MN in bipolar divisions,
11% being micronucleated without Cyt-B and 8% with
Cyt-B. In multipolar divisions, autosome laggards produced
MN more often (35%) and were mainly responsible for the
excessive MN frequency of multinucleate cells. Lagging
acentric fragments frequently formed MN, with a higher
efficiency in the presence of Cyt-B (65% bipolar, 58%
multipolar) than in its absence (41%). X chromosome
laggards were very easily micronucleated, half of them
forming MN in untreated cells and seemingly all after
Cyt-B treatment. Our findings suggest that most autosome
laggards are merely delayed in their poleward movement, eventually being engulfed by the nucleus. Lagging
fragments and X chromosomes are probably detached from
the spindle and, therefore, preferentially form MN. X
laggards are particularly efficiently micronucleated in
Cyt-B-treated cells, perhaps because they stay further away
from the poles in round cytokinesis-blocked anaphases
than in normally elongated non-blocked anaphases.
Introduction
The cytokinesis-block method for the analysis of micronuclei
(MN) (Fenech and Morley, 1985) is used both as an in vitro
test for genotoxicity and as an in vivo assay for biomonitoring
of genotoxic effects in humans. The technique is based on
the use of cytochalasin B (Cyt-B), an inhibitor of actin
polymerization, which blocks mitotic cytokinesis (cytoplasmic cleavage) but not nuclear division (Carter, 1967;
3To
MacLean-Fletcher and Pollard, 1980; Fenech and Morley,
1985). Cells that have passed through one cell division after
Cyt-B inclusion can easily be recognized by the presence of
two nuclei.
The production of binucleate cells by Cyt-B is due to
inhibition of the actin–myosin contractile ring, which in
primary human fibroblasts and some cell lines of human and
mammalian origin often appeared to be accompanied by
absence of the central spindle (Cimini et al., 1998). Although
such findings may principally reflect the intimate association
between assembly of these two structures, instead of a direct
action of Cyt-B on the spindle, presence or absence of the
spindle in the space between the poles of an anaphase–
telophase cell may influence the behaviour of laggards. Thus,
although Cyt-B has not been shown to interact directly with
mitotic spindle tubulin, Cyt-B-induced inhibition of actin
polymerization might indirectly affect separation of chromatids
and chromosome segregation.
It has clearly been demonstrated that Cyt-B-induced multinucleate human lymphocytes, which contain three, four or
more nuclei, have a very high frequency of MN, most of
which have centromeric and kinetochore signals, i.e. consist
of whole chromosomes (Lindholm et al., 1991; Norppa et al.,
1993). These MN appear to be the consequence of a very high
rate of chromosome lagging occurring in multipolar mitoses
(preceding the multinucleate state) which are produced when
cytokinesis-blocked binucleate cells further divide.
However, there is little evidence for excessive induction of
MN in the first mitosis following Cyt-B addition. Concentrations of Cyt-B inadequate to efficiently block cytokinesis
resulted in an increased frequency of micronucleated binucleate
cells, but this effect was probably due to inclusion of cells
that had remained binucleate in spite of completing two
divisions in the presence of Cyt-B and rather reflected an
increased rate of MN in the second (multipolar) than in the
first division (Zijno et al., 1994).
Several studies have detected no concentration-dependent
influence of Cyt-B on the baseline frequency of MN in
binucleate cells (Fenech and Morley, 1986; Prosser et al.,
1988; Lindholm et al., 1991; Fenech, 1993, 1997, 1998).
However, it may be argued whether the effect of Cyt-B on
MN formation can be revealed by exclusive examination of
binucleate cells, which may be considered to be affected by
Cyt-B regardless of the concentration of Cyt-B used to produce
them (Lindholm et al., 1991). If the rate of chromosome
lagging was actually higher in these cells than in less affected
cells that escape the cytokinesis block and as the proportion
of binucleate cells increases with increasing concentration of
Cyt-B (Littlefield et al., 1989; Lindholm et al., 1991), one
would expect an increase in the frequency of anaphase laggards
in bipolar anaphases with increasing concentration of Cyt-B.
In fact, lagging chromosomes appeared to become more
whom correspondence should be addressed. Tel: ⫹358 9 47472336; Fax: ⫹358 9 47472110; Email: [email protected]
© UK Environmental Mutagen Society/Oxford University Press 2002
111
G.C.-M.Falck, J.Catalán and H.Norppa
frequent in bipolar anaphases with increasing concentration of
Cyt-B, but none of the Cyt-B concentrations separately resulted
in a statistically significant increase in such laggards in
comparison with bipolar anaphases in cultures not containing
Cyt-B (Lindholm et al., 1991).
Another approach to this question is to compare MN
frequencies in binucleate and mononucleate lymphocytes
cultured without Cyt-B, taking into account that binucleate
cells contain both daughter nuclei and thus also a higher MN
frequency per cell than cells cultured in the absence of Cyt-B;
such comparisons have not suggested that Cyt-B generally
increases MN frequencies (Fenech and Morley, 1985). On the
contrary, MN frequency appears to be lowered in binucleate
cells (Channarayappa et al., 1990). Fluorescence in situ
hybridization (FISH) studies suggested that the frequency (per
1000 nuclei) of MN harbouring chromosomal fragments and
whole autosomes was lower but that of MN harbouring the X
chromosome higher in binucleate cells than in normal cells
(Norppa et al., 1993; Surrallés et al., 1996; Falck et al., 1997;
Catalán et al., 1998). Furthermore, the use of Cyt-B was
observed to reduce the induction of MN and anaphase laggards
but to increase the induction of C-anaphase and tetraploidy in
cultured human lymphocytes treated with spindle poisons
(Antoccia et al., 1993; Zijno et al., 1996; Minissi et al., 1999).
Comparison of anaphase laggards and interphase MN could
provide further information on the possible influence of CytB on MN formation. In a previous study (Catalán et al., 2000)
we observed, by FISH using X chromosome painting and a
pancentromeric DNA probe, that the X chromosome, which
is also highly over-represented in lymphocyte MN (Guttenbach
et al., 1994; Hando et al., 1994; Richard et al., 1994; Catalán
et al., 1995, 1998; Surrallés et al., 1996; Carere et al., 1999),
frequently lags behind in late anaphase of female lymphocytes
cultured in the absence of Cyt-B. The distal location of the
lagging X chromosome seemed to favour micronucleation, in
contrast to autosomes, whose more proximal placement in
anaphase–telophase cells resulted less often in MN. Here we
have studied lymphocytes of the same female donor, now with
inclusion of Cyt-B. Use of the cytokinesis block method
enabled the exclusive characterization of anaphase laggards at
the first mitosis following Cyt-B addition and MN in the
following binucleate interphase. The results were compared
with the data obtained previously without Cyt-B and the effect
of Cyt-B was also evaluated by examination of multipolar
ana-telophases and multinucleate cells.
Materials and methods
Cell cultures and treatment
Heparinized blood samples were obtained from a female donor aged 62 years.
Mononuclear leukocytes were isolated and cultured for 72 h at an initial
density of 2⫻106 cells/ml. The present experiments were performed in parallel
and identically to those reported before (Catalán et al., 2000), with the
distinction that Cyt-B (Sigma, St Louis, MO; dissolved in dimethylsulphoxide,
final concentration 6 µg/ml) was added to the cultures 44 h after phytohaemagglutinin stimulation, to obtain cytokinesis-blocked cells. In the series
performed without Cyt-B, we used a pulse treatment with 5-bromo-2⬘deoxyuridine (BrdU; Calbiochem, La Jolla, CA) 7 h before harvest for
identification of the late replicating inactive X chromosome. To obtain
comparable conditions, BrdU (final concentration 10 µg/ml) was also used in
the present series. Anaphase cells were collected using a modification
(Lindholm et al., 1991) of the technique of Ford and Congedi (1987). About
25 µl of fixed suspension was placed on wet slides and the slides were stored
in the dark at room temperature until being used for FISH.
Fluorescence in situ hybridization (FISH)
A 3 day FISH procedure was performed simultaneously with a biotin-labelled
X chromosome painting probe (1066-XB; Cambio, Cambridge, UK) and a
112
biotinylated pancentromeric probe (1141-B; Cambio) according to the manufacturer’s instructions. Slides aged for at least 3 days were fixed in acetone for
10 min at room temperature and were then, after a brief wash in 2⫻ SSC (0.3 M
NaCl, 0.03 M trisodium citrate), treated with RNase (Sigma; final concentration 100 µg/ml) for 1 h at 37°C. The slides were washed three times in 2⫻ SSC
(5 min each), treated with pepsin solution (Sigma; 50 µg/ml in 0.01 N HCl, pH
3.0), washed briefly in distilled water and dehydrated in an increasing series of
ethanol. The DNA of the cells was denatured in 70% formamide, 2⫻ SSC at
70°C for 2 min and dehydrated. The biotin-labelled X chromosome painting
probe was prewarmed at 42°C for 5 min, denatured for 10 min at 65°C and
transferred to a 37°C water bath for 60–90 min. Each slide received 15 µl of
the paint, was covered with a glass coverslip, sealed with rubber cement and
incubated overnight at 42°C in a moist chamber. The slides were then washed
at 45°C, twice with 50% formamide, 2⫻ SSC (5 min each), twice in 0.1⫻ SSC
(5 min each) and dehydrated in an increasing series of ethanol. The biotinlabelled pancentromeric probe was prewarmed at 42°C for 5 min, denatured for
10 min at 85°C and chilled on ice. Each slide received 25 µl of the probe and a
second hybridization was performed at 37°C overnight. After hybridization the
slides were washed at 37°C, once in 2⫻ SSC (5 min), twice in 50% formamide,
2⫻ SSC (5 min each), twice in 2⫻ SSC (5 min each) and once in 4⫻ SSC,
0.05% Tween-20 (5 min), followed by incubation in 4⫻ SSC, 0.5% skimmed
milk (15 min at 37°C) and a short wash in 4⫻ SSC, 0.05% Tween-20. Both
DNA probes were detected with rhodamine-conjugated Avidin D (1:50) (Vector)
and biotinylated anti-Avidin D (1:50) (Vector) antibodies. For the detection
of BrdU, mouse anti-BrdU antibody (1:50) (Becton Dickinson), fluorescein
isothiocyanate (FITC)-conjugated rabbit anti-mouse antibody (1:50) (Sigma)
and FITC-conjugated goat anti-rabbit antibody (1:50) (Sigma) were used. These
incubations were performed at 37°C for 30 min each, with three washes in
between (4⫻ SSC, 0.05% Tween-20 for 3 min at 37°C). Thereafter the slides
were dehydrated in a series of ethanol, stained in the dark for 5 min in 4⫻ SSC,
0.05% Tween-20 solution containing 5 µg/ml 4⬘,6-diamidino-2-phenylindole
(DAPI), washed in tap water, air dried and mounted in antifade solution
(Vectashield; Vector).
For the analyses of MN, the slides were hybridized with either the X
chromosome paint or the pancentromeric probe. Since the same antibodies
were used to detect both DNA probes, their presence in MN was separately
analysed. Otherwise, the FISH procedures for each probe were the same as
described above. Acetone fixation and RNase treatment as well as the last
dehydration in ethanol series were, however, left out.
Analysis of anaphase aberrations and micronuclei
The slides were scored by two microscopists under a Leitz Laborlux S
(Wetzlar, Germany) microscope equipped with epifluorescence, including filter
blocks A, I3, N2 and a triple bandpass filter (Chroma, Brattleboro, USA) for
simultaneous visualization of rhodamine (red), FITC (green), and DAPI (blue)
fluorescence (Figure 1). For the analysis of anaphases, we used the criteria of
Lindholm et al. (1991). Only cells with clearly separated poles, i.e. anaphase
B according to Ford and Congedi (1987), were scored. The cells analysed
thus represented late anaphases and early telophases; for simplicity, they are
all called anaphases in the present paper. Only cells showing both rhodamine
and FITC signals were considered for the characterization of anaphase
aberrations. The anaphases were classified as bipolar, indicating that the cell
was in its first division after Cyt-B addition, or multipolar (mostly tri- and
tetrapolar), representing second or further cell divisions.
An anaphase cell was considered aberrant if it contained lagging chromatin
or a bridge(s). The category lagging chromatin included both whole chromatids
and chromosomes as well as fragments lagging behind between the poles. A
bridge could be either a chromatid stretching between the poles or a side arm
bridge, in which the sister chromatids were still partially connected. Laggards
could represent: (i) an X chromosome, when the chromosome or the chromatid
was totally painted red (Figure 1D and E); (ii) an autosome, when it showed
a centromeric red signal (Figure 1A–C); (iii) an autosomal fragment, when
neither the red paint nor the red centromere signal was recorded (Figure 1A).
In addition, both the active and inactive X chromosomes could be distinguished
in some anaphases by considering their BrdU incorporation patterns. The late
replicating inactive X chromosome was completely labelled green, whereas
the active X was not at all or only partially green. Nevertheless, since the
lymphocyte cultures were not synchronized, distinction between the two X
homologues was not usually possible. About 2000 anaphases (1000 per scorer)
were analysed. 5000 cells were scored per cell type (i.e. binucleate and
multinucleate cells) for MN frequency and 200 MN were characterized per
probe (X chromosome and pancentromeric probe). As the pancentromeric
FISH was performed separately from X painting, C– MN contained acentric
fragments originating from both autosomes and the X chromosome. Since the
X chromosome is not known to be fragmented more often than other
chromosomes, X fragments were probably rare. Thus, they were not expected
to invalidate comparison of fragments in anaphases and in MN.
Anaphase aberrations in cytokinesis-blocked lymphocytes
Table I. Frequencies of anaphases with different numbers of poles and aberrant
anaphases among 2000 anaphases in the presence of Cyt-B (6 µg/ml,
added at 44 h) in 72 h lymphocyte cultures of a 62-year-old woman
Anaphases
Total
No.
Per centa
Aberrant
No.
Per centb
Bipolar
779
39.0
42
5.39c
Multipolar
Total
Tripolar
Tetrapolar ⬎Tetrapolar Total
291
14.6
916
45.8
14
0.70
1221
61.0
98
33.7
319
34.8
1
7.14
418
34.2
2000
460
23.0
aAmong all anaphases.
bIn each polarity class.
cIn a parallel series without
Cyt-B the frequency of aberrant bipolar anaphases
was 7.96% (Catalán et al., 2000).
Fig. 1. Characterization of late anaphase laggards in female lymphocytes.
Centromeres and X chromosomes were detected by FISH using a
biotinylated pancentromeric probe and X painting probe, respectively. The
hybridized areas were visualized using rhodamine-conjugated secondary
antibodies. (A) A bipolar aberrant anaphase with two laggards, one
autosome (red centromeric signal) and one autosomal fragment (no signal);
(B) a tripolar aberrant anaphase with two lagging autosomes; (C) a tripolar
aberrant anaphase with four lagging autosomes; (D and E) tetrapolar
anaphases each with one lagging X chromosome (uniform red staining).
All statistical comparisons were performed by the χ2 test (Statview
SE⫹Graphics v.1.03).
Results and discussion
Frequency of aberrant anaphases in the presence and absence
of Cyt-B
The results of the anaphase analysis are shown in Table I. To
accumulate 2000 anaphases for their characterization, we had
to go through almost 400 000 cells. Most of the anaphases
encountered were tetrapolar, but bipolar anaphases, cells that
were in their first division after Cyt-B addition, were also
frequent (Table I). Among the 2000 anaphases analysed in the
presence of Cyt-B, 460 (23%) were classified as aberrant. In
comparison with the parallel series without Cyt-B (Catalán
et al., 2000), there was a huge increase in aberrant anaphases
in cells treated with Cyt-B (χ2 test, P ⬍ 0.001). This effect
exclusively concerned multipolar divisions, since the
frequency of aberrant bipolar anaphases was lower (P ⬍ 0.05)
with Cyt-B (5.39%) than without it (7.96%) (Catalán et al.,
2000). In comparing these figures, it should be kept in mind
that the populations of bipolar divisions may not have been
completely identical. Bipolar anaphases that were actually
dividing for the second (or further) time in vitro were probably
more frequent in untreated cultures than in those treated with
Cyt-B, although some second division anaphases were also
likely to exist in the latter case, due to cells that had divided
for the first time before Cyt-B addition at 44 h.
The extremely high rate of anaphase aberrations in multipolar
cells is consistent with our earlier findings (Lindholm et al.,
1991), showing that further divisions of cytokinesis-blocked
binucleate lymphocytes are characterized by a high rate of
chromosome lagging and MN. This may be a general problem
of multinucleated cells, although a specific action of Cyt-B
cannot be excluded.
The frequency of anaphases was 3.4 times higher (0.54
versus 0.16%) but that of metaphases 50% lower (0.90 versus
1.88%) with Cyt-B than without it. These results suggest
(especially considering that most multipolar divisions were
equivalent to two normal divisions) that anaphases are arrested
in cytokinesis-blocked cultures, probably as a response to the
high rate of aberrations in multipolar divisions. This is in line
with our previous results (Lindholm et al., 1991) of a much
higher ratio of multipolar to bipolar anaphases than multinucleate to binucleate cells in Cyt-B-treated lymphocyte cultures. Obviously, other important factors contributing to the
latter findings are that many of the aberrant multipolar cells
die before appearing as multinucleate cells and that multipolar
divisions may produce binucleate and mononucleate cells
instead of multinucleate cells.
Type of anaphase aberrations
The aberrant anaphases were classified into those containing
(i) laggards, (ii) bridges and (iii) both laggards and bridges
(Table II). Most of the aberrant anaphases contained
laggards, which agrees with the results of the parallel series
without Cyt-B (Catalán et al., 2000) and with previous studies
(Lindholm et al., 1991; Ford and Correll, 1992). Laggards
were found in 95.2 and 93.5% of aberrant bipolar and multipolar
anaphases, respectively.
Aberrant anaphases with laggards constituted 5.1% of all
bipolar anaphases studied (Table II), which was very similar
to the frequency observed (5.3%) in the parallel series without
Cyt-B (Catalán et al., 2000). Thus, the present findings do not
support the suggestion of our previous study (Lindholm et al.,
1991) that the presence of Cyt-B increases anaphase laggards
in bipolar anaphases. Actually, no statistically significant
differences in the frequency of anaphases with laggards were
seen in the earlier study either, but there was a suggestive
dose–response effect.
113
G.C.-M.Falck, J.Catalán and H.Norppa
Table II. Different types of aberrant anaphases in cultured (72 h) female
lymphocytes in the presence of 6 µg/ml Cyt-B (added at 44 h)
Table III. Characterization of 763 anaphase aberrations in female
lymphocytes in the presence of 6 µg/ml Cyt-B (added at 44 h)
Classification
No. of aberrations in the cell
Bipolar
Multipolar
Type of aberration
FISH characterization
No. anaphases scored
No. aberrant anaphases
Laggards
One
Two
Three
Four
⬎Five
Bridges
One
Two
Three
Laggards and bridges
Laggard ⫹ bridge
2 laggards ⫹ bridge
3 laggards ⫹ bridge
Other combinations
Total no. cells with laggards
Total no. cells with bridges
Total no. laggards
779
42
1221
418
28
8
2
1
1
232
89
30
15
8
2
0
0
20
6
1
Bipolar
Multipolar
60
9 (15%)
45 (75%)
6 (10%)
2c
62
646
99 (15%)
509 (79%)
38 (6%)
55d
701
aPercentage
0
0
0
4
40 (5.13%)a
2 (0.26%)a
60 (75.2)b
6
4
4
3
391 (32%)a
44 (3.6%)a
646 (529.1)b
aFrequency
(%) of cells with each type of aberration in each polarity class
shown in parentheses.
bFrequency of laggards per 1000 cells.
Anaphases with laggards were very common among multipolar cells, 32% of which contained this aberration (Table II).
Similar high rates of laggards were earlier observed in multipolar anaphases of male lymphocyte cultures treated with
various concentrations (1.5–12 µg/ml) of Cyt-B (Lindholm
et al., 1991).
In Cyt-B-treated bipolar anaphases the frequency of cells
with bridges (0.26%) was less than one-tenth of the values
observed previously without Cyt-B (Catalán et al., 2000). In
our earlier study (Lindholm et al., 1991) we also noted that
cells with bridges were rare in bipolar anaphases with and
without Cyt-B (6 µg/ml), although frequencies ⬎1% were
seen at 1.5 and 12 µg/ml Cyt-B. At present, we do not have
an explanation as to why more bridges were found in the
parallel series without Cyt-B than with Cyt-B. Without Cyt-B,
the frequency of cells with bridges (2.8%) was only slightly
smaller than that observed in multinucleate cells (3.6%). Our
earlier data on three male donors, showing an average frequency
of 7.7% (1.5–12 µg/ml Cyt-B combined), had suggested that
the frequency of bridges is increased in multinucleate cells.
Obviously, bridges are not as typical an aberration in multinucleate cells as laggards and more studies are needed to
clarify whether they are actually increased at all.
Over one-third of the aberrant anaphases seen in the present
study contained two or more aberrations, mostly laggards.
Multiple laggards were similarly common in the parallel series
without Cyt-B (Catalán et al., 2000). Previously, simultaneous
malsegregation of more than one chromosome was observed
to be more frequent than would be expected on the basis of
independent events in binucleate human lymphocytes produced
by Cyt-B (Carere et al., 1999), suggesting the involvement of
a general malfunction of the mitotic apparatus.
FISH analysis of anaphase laggards
A closer characterization of the laggards by FISH using X
chromosome painting and a pancentromeric DNA probe,
114
Laggards
Autosomal fragments (C–X–)
Autosomes (C⫹X–)
X chromosomes (X⫹)b
Bridges (all X–)
Total
No. (%)a of anaphase aberrations
of each subtype of aberration in its aberration class and in
different polarity classes shown in parentheses.
bActivity status could be assessed for 13 X chromosomes lagging in
multipolar anaphases (10 active, 3 inactive) but for none in bipolar
anaphases.
cOne broken bridge included.
dEleven broken bridges included.
illustrated in Table III, showed that most of the laggards, in
both binucleate and multinucleate cells, regardless of Cyt-B,
were whole chromosomes, in agreement with our previous
study in men (Lindholm et al., 1991).
In bipolar cells 75% of the laggards were autosomes,
whereas acentric autosomal fragments and the X chromosome
were responsible for 15 and 10%, respectively. These figures
were similar in multipolar cells, but differed statistically
significantly (χ2 test, P ⬍ 0.01) from the percentages we
obtained in the parallel series without Cyt-B (49, 33.5 and
17.5%, respectively) (Catalán et al., 2000). In bipolar anaphases
the X chromosome represented 12% (6/51; Table III) of all
lagging chromosomes, when 4.3% (1/23) was expected by
chance. In the parallel series without Cyt-B, X overrepresentation among whole chromosome laggards was still
higher (26%, 35/133) (Catalán et al., 2000). In multipolar
anaphases the X chromosome also lagged more often than
would be expected (7%, 38/547). Taken together, the prevalence
of X chromosome lagging in Cyt-B-treated anaphases was
statistically significantly elevated (P ⬍ 0.01). Activity status
could be judged by BrdU labelling for only 13 X chromosomes
in multinucleate cells, three (23%) being considered inactive.
Although the numbers are small, they do not support
exclusive involvement of the inactive X in anaphase lagging,
in accordance with the results obtained in cultures not containing Cyt-B (Catalán et al., 2000), where both X homologues
appeared to contribute equally to X laggards. The X chromosome was not observed to be involved in anaphase bridges.
Contents of micronuclei in bi- and multinucleate cells
The results of the MN analyses are shown in Table IV and
Figure 2. The frequency of MN per 1000 cells was 11 times
higher in multinucleate cells than binucleate cells and 23-fold
in comparison with lymphocytes not treated with Cyt-B. Even
when MN frequencies in multinucleate cells were divided by
four (since most of them were supposed to contain four
diploid genomes) and those in binucleate cells by two (two
diploid genomes), multinucleate cells showed a 5.7- and
5.8-fold difference to binucleate and mononucleate cells,
respectively.
The percentages of MN harbouring fragments, autosomes
and X chromosomes were, respectively, 34.9, 22.3 and 42.9%
in binucleate cells and 19.3, 60.5 and 20.2% in multinucleate
cells. In the parallel cultures that did not contain Cyt-B (Catalán
Anaphase aberrations in cytokinesis-blocked lymphocytes
Table IV. Frequency and contents of micronuclei (MN) in cultures of
isolated female lymphocytes
Characterization
Type of cell
Mononucleate
(without Cyt-B)a
No. MN/5000 cellsb
52 (10.4)
Binucleate
(with 6 µg/ml
Cyt-B)
Multinucleate
(with 6 µg/ml
Cyt-B)
108 (21.6)
1220 (244.0)
Pancentromeric probec
No. C⫹ MN
100
No. C⫺ MN
100
Total
200
56
30
86
92
22
114
X paint probec
No. X⫹ MN
No. X– MN
Total
62
138
200
39
52
91
22
87
109
Proportion (%) of MN
Fragments
Autosomes
X chromosomes
harbouringd
50.0
19.0
31.0
34.9
22.3
42.9
19.3
60.5
20.2
aResults
on mononucleate cells are based on data published in part
previously (Catalán et al., 2000).
bFrequency for 1000 cells shown in parentheses.
cA total of 200 MN analysed per probe. Hybridization and analysis was
performed separately for the pancentromeric probe and the X paint.
dMN without pancentromeric signal were considered to contain fragments,
MN with X chromosome paint were considered to contain X chromosomes
and the remaining MN were considered to contain autosomes. Proportions
were calculated as follows: MN with fragments, percentage of C– MN; MN
with X chromosomes, percentage of X⫹ MN; MN with autosomes,
percentage of C⫹ MN minus the percentage of X⫹ MN.
et al., 2000), the respective percentages were 50.0, 19.0 and
31.0%. Thus, in comparison with untreated cells, MN in
binucleate cells contained roughly equal proportions of
autosomes, a 0.7 times smaller proportion of fragments and
a 1.4-fold proportion of X chromosomes. The difference in
MN contents between cytokinesis-blocked binucleate cells and
untreated cultures was statistically significant (χ2 test, P ⬍
0.05) for fragments and X chromosomes.
Comparison of anaphase laggards and micronuclei
Figure 2 shows the frequencies of various types of laggards
and MN, as adjusted according to the number of diploid
genomes in each cell type, to allow direct comparison. Frequencies obtained from bipolar anaphases and binucleate interphases
were divided by two and those from multipolar anaphases and
multinuclear cells by four.
It is evident that the high MN frequency in multinucleate cells
was primarily due to autosomes, with a smaller contribution by
fragments and X chromosomes. This mainly resulted from the
very high anaphase lagging of autosomes, rather than from
their preferential micronucleation, since 35% of them formed
MN. Autosome laggards were very poorly micronucleated in
bipolar divisions with (8%) and without (11%) Cyt-B, clearly
less efficiently than lagging X chromosomes and fragments.
The inefficient micronucleation of autosomes may reflect
the fact that autosome laggards are relatively close to the poles
(Catalán et al. 2000) and may, therefore, be easily included in
the main nuclei. The proximal location of autosome laggards
actually suggests that many of them are not totally detached
from the spindle but are just delayed in their movement, finally
reaching the pole without forming MN. Lack of the central
spindle (Cimini et al., 1998) in the presence of Cyt-B might
Fig. 2. Frequency of anaphase laggards and micronuclei (MN) in cultured
(72 h) female lymphocytes after a 28 h in vitro treatment (started at 44 h)
with Cyt-B (6 µg/ml). Centromeres and X chromosomes were detected by
FISH. Results are shown separately for fragments, autosomes and X
chromosomes in (A) untreated bipolar anaphases and mononucleate cells
(Catalán et al., 2000), (B) Cyt-B-treated bipolar anaphases and binucleate cells
and (C) multipolar anaphases and multinucleate cells. The original frequencies
of anaphase laggards were modified to facilitate comparison among different
cell types. Frequencies for bipolar anaphases and binucleate cells were divided
by two and those for multipolar anaphases and multinucleate cells by four.
115
G.C.-M.Falck, J.Catalán and H.Norppa
enhance such a delay, which could explain the higher rate
of autosome laggards in bipolar divisions with Cyt-B than
without it.
We observed earlier that the frequency of autosomecontaining MN (per 1000 nuclei) was lower in binucleate cells
than in untreated cells (Surrallés et al., 1996; Catalán et al.,
1998). This agreed with our previous suggestion that the
distance between spindle poles may be shorter in cytokinesisblocked than normal cells, thus favouring laggard inclusion
within the daughter nuclei in binucleate cells (Norppa et al.,
1993; Surrallés et al., 1996; Falck et al., 1997). Minissi et al.
(1999) subsequently observed that the pole-to-pole distance in
anaphase lymphocytes is reduced in the presence of Cyt-B.
Thus, centrally located laggards may be nearer to the poles
(and more easily engulfed in the nuclei) in round cytokinesisblocked bipolar anaphases than in elongated non-blocked
anaphases. The present study did not clarify this question,
since there was no difference in MN frequency between
cytokinesis-blocked binucleate cells and untreated cells.
X laggards generally formed MN efficiently, 49% of them
being micronucleated without Cyt-B (Figure 2). In cells treated
with Cyt-B frequencies were actually higher for X-positive
MN than for X laggards, suggesting that all X laggards are
micronucleated. The high MN to laggard ratios may also
reflect the fact that MN are easier to detect than anaphase
laggards. In untreated lymphocytes lagging X chromosomes
often consisted of both sister chromatids and tended to be
further away from the poles than lagging autosomes (Catalán
et al., 2000). X laggards are distally located and easily form
MN, probably because they are not attached to the spindle.
This may be due to a failure in microtubule attachment, since
most X-positive MN were observed to be kinetochore-negative
(Hando et al., 1994). Furthermore, loss of the inactive X from
the nucleus should be more easily tolerated than loss of
autosomes.
In round cytokinesis-blocked bipolar anaphases with no
cytokinesis and no central spindle, X laggards may actually
stay in the periphery of the cell further away from the poles
than in normally elongated anaphases of the same cytoplasmic
volume, where constriction during cytokinesis and the central
spindle may more easily drive them to the nuclei. These factors
could contribute to the exaggerated micronucleation of the X
chromosome in cytokinesis-blocked cells and might explain
earlier observations of higher frequencies of X-positive MN
in binucleate than untreated cells (Surrallés et al., 1996;
Catalán et al., 1998). In the present study also, the frequency
of X-positive MN was higher in binucleate than in mononucleate cells, despite the opposite situation with X laggards.
Lagging acentric fragments also effectively formed MN
(41% without Cyt-B, 65% in bipolar divisions with Cyt-B and
58% in multipolar divisions). This could be expected, since
acentric fragments are not attached to the spindle. A decrease
in the frequency of MN with fragments has previously been
seen in binucleate cells as compared with untreated cells
(Norppa et al., 1993; Surrallés et al., 1996; Falck et al., 1997;
Catalán et al., 1998). Our results suggest that this is due to
less lagging behind of fragments in bipolar anaphases in the
presence of Cyt-B than in its absence, rather than inefficient
micronucleation of lagging fragments. It is presently unclear
how Cyt-B could affect fragment lagging.
Conclusions
We found that autosome lagging is the major anaphase aberration observed in multipolar division of female lymphocytes in
116
the presence of Cyt-B (6 µg/ml). Subsequently, most ‘extra’
MN seen in multinucleate cells contain autosomes. In lymphocyte cultures treated with Cyt-B, bipolar anaphases appear to
show more autosome laggards but fewer lagging fragments
and X chromosomes than without Cyt-B. However, autosome
laggards form MN very inefficiently in bipolar divisions,
possibly because many of them are still attached to the spindle.
On the other hand, most X laggards are probably detached
from the spindle and are located in the periphery of the cell.
In the presence of Cyt-B all lagging X chromosomes appear
to form MN, and half of X laggards also do so in untreated
cells. The differential effectivity of X chromosome micronucleation with and without Cyt-B may explain why binucleate
cells tend to show increased frequencies of X-positive MN.
The findings may be partly explained considering that the
distally located lagging whole X chromosomes stay further
away from the poles in round anaphases of cytokinesis-blocked
cells than in elongated anaphases of non-blocked cells. The
lowered frequency in binucleate cells of MN harbouring
acentric fragments would appear to reflect infrequent anaphase
lagging of such fragments in Cyt-B-treated bipolar anaphases.
Acknowledgements
We thank Dr C.Moreno for help with statistics, Dr J.Surrallés for advice on
the labelling of the late replicating X chromosome and Ms Hilkka Järventaus
for technical assistance. G.C.-M.F. received financial support from the Finnish
Work Environment Fund.
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Received on June 22, 2001; accepted on October 5, 2001
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