Mutagenesis, Vol. 17, No. 2, 111-117,
March 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
Nature of anaphase laggards and micronuclei in female cytokinesis-blocked lymphocytes
1 Laboratory 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 2 Department of Anatomy, Embryology and Genetics, University of Zaragoza, Zaragoza, Spain
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Abstract |
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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.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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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; 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 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 Cyt-B 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.
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Materials and methods |
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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 2x106 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 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 2x 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 2x 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, 2x 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, 2x SSC (5 min each), twice in 0.1x SSC (5 min each) and dehydrated in an increasing series of ethanol. The biotin-labelled 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 2x SSC (5 min), twice in 50% formamide, 2x SSC (5 min each), twice in 2x SSC (5 min each) and once in 4x SSC, 0.05% Tween-20 (5 min), followed by incubation in 4x SSC, 0.5% skimmed milk (15 min at 37°C) and a short wash in 4x 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 (4x 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 4x 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.
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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.
All statistical comparisons were performed by the 
2 test (Statview SE+Graphics v.1.03).
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Results and discussion |
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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 (2 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.
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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.
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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.
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, 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).
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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 (
2 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 over-representation 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.
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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 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 (2 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 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 cytokinesis-blocked 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 cytokinesis-blocked 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 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.
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Acknowledgments |
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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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Notes |
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3 To whom correspondence should be addressed. Tel: +358 9 47472336; Fax: +358 9 47472110; Email: hannu.norppa{at}occuphealth.fi
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References |
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Received on June 22, 2001; accepted on October 5, 2001.
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