Mutagenesis, Vol. 17, No. 4, 337-344,
July 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
Elimination of micronucleated cells by apoptosis after treatment with inhibitors of microtubules
1 Laboratory for Cell Genetics, Free University of Brussels, Pleinlaan 2, B-1050 Brussels, Belgium and 2 Centro di Genetica Evoluzionistica CNR, Via degli Apuli 4, I-00185 Rome, Italy
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Abstract |
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Two major mechanisms responsible for chromosome segregation errors are non-disjunction and chromosome loss, both leading to aneuploidy. Previous studies in our laboratory showed the existence of thresholds for the induction of chromosome non-disjunction and chromosome loss and the induction of apoptosis by microtubule inhibitors. From a mechanistic point of view one can expect that apoptosis contributes to the elimination of cells with premutagenic/mutagenic lesions. If aneuploid cells were eliminated by the induction of apoptosis below the threshold concentrations for chromosome loss and non-disjunction, the defined thresholds would not be applicable to cells unable to undergo apoptosis. The aim of this study was to investigate whether apoptosis was induced directly or indirectly as a response to aberrant chromosome segregation below the thresholds for the induction of chromosome loss and non-disjunction, as previously defined by us. Therefore, human lymphocytes were exposed in vitro to five concentrations of nocodazole and five concentrations of carbendazim representing the threshold concentrations for chromosome non-disjunction and chromosome loss, two concentrations below the lowest threshold and one concentration between the two threshold values. After 48 h exposure to the aneugens, induction of apoptosis was analysed by the annexin-V test. The frequencies of chromosome non-disjunction and chromosome loss were estimated in cytokinesis-blocked human lymphocytes in combination with FISH; this methodology was applied to whole cell cultures as well as to apoptotic and viable cell fractions obtained using magnetic annexin microbead cell sorting. Our results suggest that elimination of aneuploid cells does occur. However, the efficiency of disappearance of micronucleated cells is higher than for cells presenting chromosome non-disjunction. The correlation found between early apoptotic events and micronucleus formation could account, at least in part, for the specific elimination of aneuploid cells.
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Introduction |
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Deficiencies in or impairment of the different structures involved in accurate segregation and migration of sister chromatids may lead to an important genomic imbalance. Two important mechanisms responsible for chromosome segregation errors are non-disjunction and chromosome loss, both leading to aneuploidy. Chromosome non-disjunction occurs when chromatids do not separate normally but stick together and move to one pole during anaphase, resulting in two daughter cells containing respectively 2n + x and 2n – x chromosomes at the next mitosis. Chromosome loss may be the result of either non-attachment of the chromosome kinetochore to the spindle microtubules or chromosome lagging at anaphase and gives rise to a micronucleus. It is widely recognized that aneuploidy is an integral component in the development of human tumours and the acquisition of malignancy (for a review see Duesberg and Rasnick, 2000
). Several studies in rodents also showed that chemical exposure leads to the induction of specific aneuploidies, which in their turn play a role in tumour progression (Oshimura and Barrett, 1986; Haesen et al., 1993; Van Goethem et al., 1995).
Previous studies in our laboratory showed the existence of thresholds for the induction of chromosome non-disjunction and chromosome loss by microtubule inhibitors such as nocodazole, a chemotherapeutic drug, and carbendazim, a fungicide (Elhajouji et al., 1995, 1997). The threshold characteristics for chromosome non-disjunction induced by carbendazim were confirmed by Bentley et al.(2000) using chromosome-specific centromeric probes for chromosomes 1, 8, 11, 17, 18 and X.
Furthermore, it was shown that the mitotic spindle inhibitor nocodazole is able to trigger apoptosis (Casenghi et al., 1999; Verdoodt et al., 1999). Apoptosis is a regulated form of cell death essential in development and tissue homeostasis; this active cell suicide mechanism is characterized by shrinkage of cells, segmentation of the nucleus, fragmentation of the cytoplasm into membrane-bound apoptotic bodies and condensation and cleavage of DNA, followed by internucleosomal degradation (Saikumar et al., 1999).
From a mechanistic point of view one can expect that apoptosis contributes to the elimination of cells with premutagenic/mutagenic lesions. If apoptosis is induced below the threshold concentrations for chromosome loss and non-disjunction, resulting in the elimination of aneuploid cells, the defined thresholds would not be applicable to cells unable to undergo apoptosis.
The aim of this study was to investigate whether apoptosis was induced directly or indirectly as a response to aberrant chromosome segregation below the thresholds for the induction of chromosome loss and non-disjunction, defined by us earlier (Elhajouji et al., 1995, 1997). Therefore, human lymphocytes were exposed in vitro to five concentrations of nocodazole and five concentrations of carbendazim: the threshold concentrations for chromosome non-disjunction and chromosome loss, two concentrations below the lowest threshold and one concentration between the two threshold values. The choice of these concentrations was based on our previous studies on threshold values for the two aneugens. After 72 h phytohaemagglutinin (PHA) stimulation, corresponding to 48 h exposure to the aneugens, induction of apoptosis was analysed with annexin-V. The frequencies of chromosome non-disjunction and chromosome loss were estimated in cytokinesis-blocked human lymphocytes in combination with FISH. In order to check whether aneuploid cells are preferentially driven to apoptosis, we sorted apoptotic and viable cells from the same cultures using magnetic annexin beads and measured micronucleus induction, chromosome loss (MNCen+) and chromosome non-disjunction in the apoptotic and viable fractions.
Our results suggest that elimination of micronucleated cells or cells with chromosome non-disjunction does occur. The frequency of micronucleated cells was higher in the apoptotic fraction than in the viable fraction. Cells bearing non-disjunction seemed to be a less strong trigger for apoptosis. The results obtained suggest that the presence of micronuclei (MN) in a cell correlates with apoptosis and therefore contributes to the elimination of aneuploid cells.
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Methods and materials |
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Cell cultures and treatment
Human peripheral blood samples were obtained from a healthy female donor of <25 years of age. Lymphocytes were isolated using Ficoll-Pacque (Pharmacia Biotech, Uppsala, Sweden) and were cultured in Ham's F-10 medium containing 25 mM L-glutamine (Gibco BRL, Paisley, UK), supplemented with 15% foetal calf serum (Gibco BRL) and 2% PHA A 16 (Murex Biotech, Dartford, UK) and incubated in 5% CO2 in a humidified incubator at 37°C.
Nocodazole (Acros Organics, Belgium) and carbendazim (Aldrich Chemie, Steinheim, Germany) were dissolved in dimethylsulphoxide (DMSO) (Merck, Darmstadt, Germany). The control cultures were treated with 0.5% DMSO.
After 24 h PHA stimulation, two parallel lymphocyte cultures were exposed to one concentration of the studied aneugens and one culture to DMSO as a control. Forty-four hours after stimulation, cytochalasin B (Sigma Chemical Co., Belgium) was added at 6 µg/ml to the cultures to block cytokinesis and to obtain binucleated cells. After 72 h PHA stimulation, corresponding to 48 h exposure to the aneugens, 1 ml of each culture was used to determine the frequency of apoptotic, necrotic and viable cells with annexin-V and another 1 ml of the cultures was fixed to prepare slides for FISH analysis. The remaining 5 ml was subjected to isolation of apoptotic and non-apoptotic (viable) cells, using magnetic annexin microbead cell sorting.
After separation, the two cell fractions were fixed and placed on slides for FISH analysis. Prior to fixation cells were subjected to a cold hypotonic treatment (0.075 M KCl), then immediately centrifuged and fixed three times with fixative (methanol:acidic acid, 3:1). The fixed cells were dropped onto slides using Pasteur pipettes, air dried and stored at –20°C (Elhajouji et al., 1997). This protocol was followed in 10 experiments where five concentrations of nocodazole and five concentrations of carbendazim were analysed: the threshold concentrations for chromosome non-disjunction and chromosome loss, two concentrations below the lowest threshold and one concentration between the two thresholds.
Annexin-V staining
Cells were collected by centrifugation and resuspended in 100 µl of annexin labelling solution consisting of 2% annexin-V-FLUOS (Roche Diagnostics, Belgium) and 0.1 µg/ml propidium iodide (PI) (Sigma Chemical Co.) in HEPES buffer containing 10 mM HEPES (Sigma Chemical Co.), 140 mM NaCl, 2 mM CaCl2, 5 mM KCl and 1 mM MgCl2, pH 7.4. Cells were incubated in the dark in this solution for 15 min and dropped onto slides. Cell preparations were analysed with a Zeiss Axioscop fluorescence microscope (Carl Zeiss, Oberkochen, Germany), equipped with double bandpass filter no. 24 (Zeiss) to visualize the annexin-V-FLUOS-labelled apoptotic cells and the necrotic cells stained with PI, at a magnification of 400x. The viable cells were analysed in parallel with normal transmitted light. 1000 cells per culture were scored.
Magnetic annexin microbead cell sorting
Cells were collected by centrifugation and resuspended in 80 µl of 1x Binding Buffer and 20 µl of MACS Annexin-V MicroBeads, both from an Apoptotic Cell Isolation Kit (Miltenyi Biotec GmbH, Germany). Cells were incubated for 15 min at 6–12°C. After washing the cells with 1x Binding Buffer, the cell suspension was applied to a LS+ column (Miltenyi Biotec GmbH). The column was fixed in a separator in a magnetic field. The negative (viable) cells were passed through the column. After rinsing with 1x Binding Buffer, the column was removed from the separator, placed on a collection tube and, after adding 1x Binding Buffer, the positive (apoptotic) cell fraction was flushed out using a plunger. After separation and collection of the viable and apoptotic cells, the cell suspensions were immediately fixed for FISH analysis with a pancentromeric probe and centromeric chromosome-specific probes for chromosomes 1 and 17. Cells and solutions were kept cold to avoid rapid degradation.
FISH with a pancentromeric probe
FISH with a pancentromeric probe was carried out with a 30 nt synthetic oligomer (SO-
AllCen, synthetic oligomer- all centromeres) which hybridizes to the conserved region of the -satellite DNA present at centromeres of all human chromosomes (Meyne et al., 1989). The probe was 3'-end-labelled by terminal deoxynucleotidyl transferase (Gibco BRL, Grand Island, NY) with biotin-16-dUTP (Roche Diagnostics, Belgium). The cells were pretreated with pepsin (Sigma Chemical Co.) (0.005% in 10 mM HCl) for 10 min. Cells and probe were denatured simultaneously at 80°C for 3–4 min. Following overnight hybridization, immunofluorescence detection of the probe was performed using avidin–FITC (Vector Laboratories, Burlingame, CA) and biotinylated goat anti-avidin (Vector Laboratories). After dehydratation in a graded ethanol series, the slides were counterstained with ethidium bromide (5 µg/ml) in a DABCO-antifade solution (Sigma Chemical Co.).
FISH with centromeric chromosome-specific probes
FISH with centromeric chromosome-specific probes was performed with the pUC1.77 probe (Cooke and Hindley, 1979), which hybridizes to the satellite II pericentromeric region of chromosome 1, and the D17Z1 probe (ATTC, Rockville, MD), to label the centromere of chromosome 17. The probes were labelled with digoxigenin-11-dUTP (Roche Diagnostics) or biotin-11-dUTP (Roche Diagnostics) by nick translation according to the instructions of the suppliers (Life Technologies BRL, Paisley, UK). FISH was essentially carried out as described by Viegas-Pequinot et al. (1989). Slides were treated with 0.05% RNase in 2x SSC (0.3 M sodium chloride, 0.03 M sodium citrate) for 60 min at 37°C, rinsed in 2x SSC, then treated with 0.005% pepsin in 10 mM HCl for 10 min at 37°C and dehydrated in a graded ethanol series (50, 75 and 100%). Slides and probes were denatured simultaneously for 4 min at 90°C. Hybridization took place overnight at 37°C. After hybridization, slides were washed twice for 7 min in 2x SSC, 50% formamide and twice for 2 min in 2x SSC, both at 43°C. Before immunofluorescence detection of the probes, the slides were rinsed in blocking buffer (0.5% blocking reagent in 4x SSC; Roche Diagnostics). The slides were then incubated with avidin–FITC (Vector Laboratories), then with monoclonal mouse anti-digoxigenin (Roche Diagnostics) together with a biotinylated goat anti-avidin antibody (Vector Laboratories) and finally with avidin–FITC (Vector Laboratories) and with Texas Red-conjugated sheep anti-mouse antibody (Amersham, Little Chalfont, UK). After dehydratation in a graded ethanol series, the cells were counterstained with DAPI (Roche Diagnostics) in a DABCO-antifade solution (Sigma Chemical Co.).
FISH analysis
For FISH analysis ~1000 cytochalasin B-blocked binucleated cells (CB) were scored per culture and per concentration, with a maximum of 1000 CB per culture. The MN in binucleated lymphocytes were examined for the presence of one or more spots and were classified as centromere-positive (MNCen+) or centromere-negative (MNCen-), the latter showing no centromeres (Elhajouji et al., 1997; Kirsch-Volders, 1997). The standard scoring criteria for MN (1/3 diameter, no overlap, shape) were used (Kirsch-Volders et al., 2000). For chromosomal non-disjunction, the scoring was restricted to binucleated cells having the diploid number for the chromosomes analysed (two spots for each of the two probes). The distribution of the signals for both probes between the binucleated cells was scored as 2/2, 1/3 and 0/4. The events involving chromosome 1 were scored independently of those involving chromosome 17 but recorded in parallel per cell.
The preparations were examined with a Zeiss Axiscop microscope (Carl Zeiss) equipped with triple bandpass filter no. 25 (Zeiss) to visualize the FITC/Texas Red-labelled probe and the orange-red ethidium or DAPI counterstaining, at a magnification of 400x.
Statistical analysis
For statistical analysis of the differences between treated and control cultures the 
2 test was applied.
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Results |
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Induction of apoptosis
The effects of one concentration of nocodazole or carbendazim were studied in each experiment. After 24 h PHA stimulation, two parallel lymphocyte cultures (all from the same donor) were exposed to one concentration of the studied aneugens and one culture to DMSO as a control. After 72 h PHA stimulation, corresponding to 48 h exposure to the aneugens, the frequency of apoptotic cells was determined by the annexin-V test before fractionation. FISH was applied to assess chromosome loss and non-disjunction before and after fractionation of apoptotic and viable cells.
Since a large amount of blood was required to test one concentration, it was impossible to analyse all the concentrations of the two compounds simultaneously. Therefore, in the analysis of each concentration tested, a comparison was always made with the control culture prepared and analysed in the same experiment.
To assess the induction of apoptosis by the annexin-V test two cultures treated in parallel and one control culture were analysed. In the early stages of apoptosis translocation of phosphatidylserine (PS) occurs from the inner part of the plasma membrane to the outer layer. Annexin-V has a high affinity for PS and is therefore used to detect early apoptotic cells. Since necrotic cells also expose PS due to loss of membrane integrity, PI was used simultaneously to discriminate necrotic cells from annexin-V-positive early apoptotic cells. Only early apoptotic cells (annexin-V-positive, PI-negative) were taken into account. No statistically significant difference in the frequency of apoptotic cells was observed between the two treated cultures, therefore, their mean was taken for comparison with the control culture. Both compounds induced a statistically significant increase (P < 0.001) in apoptotic cells at all concentrations tested. This increase was not dose dependent (Figure 1).
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Induction of chromosome loss
Before fractionation. To evaluate the frequencies of chromosome loss, the in vitro micronucleus assay was combined with FISH analysis for pancentromeric regions. The MN in binucleated lymphocytes (MNCB) were examined for the presence of one or more centromeric spots and were classified as MNCen+ or MNCen-. First, the MN frequencies in MNCB and the frequencies of chromosome loss (MNCen+) were scored in the lymphocyte cultures before fractionation on columns, to assess whether the selected concentrations were indeed situated under the threshold concentrations for chromosome loss for carbendazim and nocodazole, respectively, in the studied donor. Two cultures were prepared for each concentration of carbendazim or nocodazole and the mean of the two exposed cultures was taken to compare with the controls and with the other concentrations of the two aneugens studied. If possible, 1000 binucleated cells were scored per culture. However, in the apoptotic fractions this was not always the case, since the percentage of apoptotic cells was relatively low (between 10.4 and 20.1%) in all the cultures analysed.
No concentration-dependent increase in MNCB was observed in the presence of carbendazim. For all the tested concentrations of carbendaziman an increase in MNCB was observed compared with the controls, but this increase was only statistically significant for 2.65 µM (P > 0.05), a concentration situated above the threshold. As far as chromosome loss is concerned, the two concentrations (2.65 and 2.847 µM) which induced a statistically significant increase in MNCen+ (P < 0.05) are situated above the threshold. Below the threshold concentration for chromosome loss (0.523 and 1.5 µM) and at the threshold concentration of 2.47 µM the majority of micronucleated cells were MNCen- (range 61.92–63.33%). Above this threshold, in contrast, most of MNCBs (70.84–83.33%) were MNcen+, as expected from a good chromosome loss inducer such as carbendazim (Table IA).
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In the presence of nocodazole, which is a weaker inducer of chromosome loss as compared with carbendazim, no statistically significant increase in MNCB frequency was recorded, even if at the highest concentration tested almost 50% of MNCBs were MNCen+ (Table IB
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After fractionation. The overall frequencies of apoptotic cells analysed by the annexin-V test were between 15.9 and 19.1% for carbendazim and between 16 and 20.1% nocodazole.
After separation of viable and apoptotic lymphocytes, FISH for pancentromeric regions was applied to the two cell fractions obtained (the viable and apoptotic cell fractions). Tables II and III give the frequencies of MNCB and the frequencies of MNCen- and MNCen+ cells induced by carbendazim and nocodazole, respectively, in the apoptotic (Tables IIA and IIIA) and viable cell fractions (Tables IIB and IIIB). In order to compare directly micronucleated cells in apoptotic versus viable cell fractions, we expressed the data as the ratio between apoptotic and viable cells. A ratio higher than one indicates a preferential presence of micronucleated cells among apoptotic cells.
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As shown in Figure 2
, the frequency of MNCBs was significantly increased in the fraction containing apoptotic cells, corresponding to selective sorting of micronucleated lymphocytes in the control and treated cultures for the two compounds. Micronucleation per se seems to constitute a strong apoptotic signal, where untreated micronucleated cells significantly concentrated in the apoptotic fraction (Figure 2).
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Chromosome loss was measured by determining MNCen+. As shown in Figure 3A and B
, when MNcen+ are taken into account, the observed phenomenon of accumulation of micronucleated cells in the apoptotic fraction is confirmed and, interestingly, peaks at the concentrations corresponding to the non-disjunction threshold for both compounds.
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Induction of non-disjunction
Before fractionation. Frequencies of chromosome non-disjunction were estimated in cytokinesis-blocked binucleated lymphocytes in combination with FISH using chromosome-specific pericentromeric probes for chromosomes 1 and 17. First, the frequencies of chromosome non-disjunction between the two daughter nuclei were scored in the lymphocytes before fractionation on the columns, to assess whether the selected concentrations are situated below the threshold concentrations for chromosome non-disjunction for carbendazim and nocodazole, respectively, in the donor studied. Two cultures were prepared for each concentration of carbendazim and nocodazole; the mean of the two exposed cultures was taken for comparison with the controls and with the other concentrations of the two aneugens studied. Again, if possible 1000 binucleated cells were scored per culture.
To estimate chromosomal non-disjunction frequencies in the total genome, assuming that non-disjunction occurs randomly over the chromosomes, the initial frequencies observed for chromosomes 1 and 17 were multiplied by 23/2.
Table IA and B
shows the frequencies of chromosome non-disjunction observed for carbendazim and nocodazole, respectively, at the concentrations used. As expected, for both compounds highly statistically significant increases were observed at and above the threshold values.
After fractionation.
After fractionation into viable and apoptotic cell fractions, the frequencies of chromosome non-disjunction were estimated in the two cell fractions obtained (viable and apoptotic) using FISH with pericentromeric probes for chromosomes 1 and 17 (Tables IV and V).
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As described before for chromosome loss, non-disjunction events seemed to be more concentrated among apoptotic than viable cells. However, in the case of non-disjunction, this effect was less evident as compared with MN. It can be observed that for all the concentrations tested the ratio between apoptotic and viable cells is close to one and similar for all the concentrations used (Figure 4
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Discussion |
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Previous studies in our laboratory showed the existence of thresholds for the induction of chromosome non-disjunctiona and chromosome loss by microtubule inhibitors such as carbendazim, a pesticide, and nocodazole, a chemotherapeutic drug (Elhajouji et al., 1995
, 1997). The existence of these thresholds may have important implications for hazard and risk assessment. Therefore, the aim of this work was to assess whether apoptosis led to the elimination of aneuploid cells induced by two aneugens, carbendazim and nocodazole, below the statistically defined thresholds (Elhajouji et al., 1995, 1997). If this was the case, the defined thresholds would not be applicable to, for example, premalignant cells unable to undergo apoptosis and to individuals deficient for some apoptosis-related pathways. Protecting these genetically susceptible persons would imply that the recommended thresholds should be lowered to concentrations at which they do not show a significant increase in the background frequencies of mutations. Similar considerations should be applied in chemotherapy for the estimation of treatment dose.
Due to the high specificity of nocodazole and carbendazim for their molecular target (Arg390 of ß-tubulin), one may assume that there is no other target and that all toxic effects observed are related to this specific interaction with microtubules. In order to avoid artefacts, e.g. scoring of MN induced before the treatment, we used cytochalasin B to arrest cytokinesis. This protocol allowed the assessment of aneuploidy strictly in binucleates and to identify as binucleates those cells which divided once in vitro and to score non-disjunction in cells where the sum of the two macronuclei corresponded to a balanced diploidy. Moreover, the combination of FISH and magnetic sorting of apoptotic versus viable cells proved to be adequate to analyse whether chromosome loss or non-disjunction could themselves trigger apoptosis. Since annexin labelling is based on an early feature of apoptosis, we obtained cells which had not yet lost their integrity and were therefore suitable for the application of FISH. Furthermore, we can assume that the cells which were annexin-positive, indicating externalization of PS as an early feature of apoptosis, were really eliminated by apoptosis and underwent the whole process of apoptosis until ultimate degradation. This is confirmed by previous studies in our laboratory (Verdoodt et al., 1999), where apoptotic cells induced by nocodazole were detectable by TUNEL and Giemsa staining, two methodologies which detect later stages of apoptosis (chromatin condensation and DNA fragmentation, respectively). We verified that the sorting procedure with magnetic beads was highly efficient and sensitive. After sorting, indeed, ~80% of the cells in the apoptotic fraction (range 75.4–91.7%) were annexin-V-positive, i.e. early apoptotic cells. This avoided contamination with late apoptotic and necrotic cells which would no longer have been stainable for FISH analysis.
A first objective in this study was to assess whether carbendazim and nocodazole can induce apoptosis at concentrations below or close to the threshold for induction of aneuploidy. A statistically significant increase in apoptosis is induced for both carbendazim and nocodazole at all concentrations tested, including those below the threshold values for chromosome loss (MNCen+) and non-disjunction, respectively. However, no concentration-dependent induction of apoptosis was found, neither for carbendzim nor for nocodazole. In the present study the concentrations used were very close to each other, which could explain the absence of a dose-dependent induction of apoptosis.
The second objective was to identify the presence of aneuploid cells (microncleated or with chromosome malsegregation) in viable versus apoptotic cells. The selective sorting of apoptotic cells with micronucleated cells in both control and treated cultures suggests that MN (in general, meaning both MNCen+ and MNCen-) can constitute a signal for apoptosis and that MN formation is related to the early stages of apoptosis. To elucidate how MN trigger apoptosis more information is needed about the specific constitution and origin of the MN. Therefore, in future experiments more mutagens, inducing different adducts/lesions, should be tested to assess which type(s) of MN resulting from a specific lesion induces apoptosis. The high ratio of spontaneously induced micronucleated cells in the apoptotic fraction of the controls confirms the apoptogenic nature of MN.
It has been reported that MN formation corresponds to activation of important signalling proteins, such as the tumor suppressor p53 (Sablina et al., 1998). The nature of the damage induced by both aneugens below the thresholds for non-disjunction could be qualitatively different from that induced above the threshold (Wilson and Jordan, 1995), e.g. interphase disruption of microtubules (Verdoodt et al., 1999). It is known that important regulatory proteins such as p53 are actively transported to different cellular compartments through interphase microtubules (Giannakakou et al., 2000; Ciciarello et al., 2001). Thus, interphase microtubules seem to play an important role in signal transduction. Low doses of microtubule inhibitors may impair the cellular ability to activate apoptosis.
Cells presenting non-disjunction versus euploid cells were found in approximately the same relative proportions in both the viable and apoptotic cell fractions and no threshold effect was detected. This suggests that non-disjunctional events are well tolerated. Hence, the probability of a cell bearing a gain or loss of a chromosome surviving is high. Moreover, this confirms our suggestion that factors other than aneugenic events may contribute to the induction of apoptosis after treatment with carbendazim and nocodazole.
However, it cannot be excluded that a non-disjunction fails to resolve itself and results in chromosome loss or, vice versa, that chromosome loss may be resolved by random incorporation into a daughter nucleus.
A very striking result of the present study is that, for both compounds and despite the different abilities to induce either non-disjunction and chromosome loss and the differences in the threshold values for non-disjunction and chromosome loss, the highest probability of a micronucleated cell being apoptotic was found at the threshold concentration for non-disjunction. This suggests that most of the MN induced at that aneugenic concentration disappear due to apoptosis. Therefore, the threshold values calculated for chromosome loss could be greatly overestimated.
From our data we can conclude that elimination of micronucleated cells or cells with chromosome non-disjunction does occur. However, the trigger for disappearance of these cells is most probably not aneuploidy but the presence of MN. At the same time, a role of microtubule depolymerization during interphase cannot be excluded. These cells result from abnormal mitotic segregation in the first mitosis allowing further cell progression in interphase 2, but would be eliminated by chance because of further interaction of the microtubule inhibitors with the interphase cytoskeleton.
In the future, analysis of the induction of aneuploidy in cell lines deficient for apoptosis-related genes such as p53, bcl-2/bax or caspase-3 would provide additional arguments to identify the major pathways responsible for induction of apoptosis and the relation with aneuploidy after exposure to tubulin inhibitors.
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Acknowledgments |
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This work was supported by the EU research programmes ENV4-CT97-0471 and QLK4-CT-2000-00058.
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Notes |
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2 To whom correspondence should be addressed. Tel: +32 2 629 34 27; Fax: +32 2 629 27 59; Email: idecordi{at}vub.ac.be
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References |
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Received on December 19, 2001;
revised on March 21, 2002;
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  J. Muller, I. Decordier, P. H. Hoet, N. Lombaert, L. Thomassen, F. Huaux, D. Lison, and M. Kirsch-Volders Clastogenic and aneugenic effects of multi-wall carbon nanotubes in epithelial cells Carcinogenesis, February 1, 2008; 29(2): 427 - 433. [Abstract] [Full Text] [PDF]
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S. Bonassi, A. Znaor, M. Ceppi, C. Lando, W. P. Chang, N. Holland, M. Kirsch-Volders, E. Zeiger, S. Ban, R. Barale, et al. An increased micronucleus frequency in peripheral blood lymphocytes predicts the risk of cancer in humans Carcinogenesis, March 1, 2007; 28(3): 625 - 631. [Abstract] [Full Text] [PDF]
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S. Fukushima, A. Kinoshita, R. Puatanachokchai, M. Kushida, H. Wanibuchi, and K. Morimura Hormesis and dose-response-mediated mechanisms in carcinogenesis: evidence for a threshold in carcinogenicity of non-genotoxic carcinogens Carcinogenesis, November 1, 2005; 26(11): 1835 - 1845. [Abstract] [Full Text] [PDF]
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 I. Decordier, E. Cundari, and M. Kirsch-Volders Influence of caspase activity on micronuclei detection: a possible role for caspase-3 in micronucleation Mutagenesis, May 1, 2005; 20(3): 173 - 179. [Abstract] [Full Text] [PDF]
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F. Sun, I. Betzendahl, F. Pacchierotti, R. Ranaldi, J. Smitz, R. Cortvrindt, and U. Eichenlaub-Ritter Aneuploidy in mouse metaphase II oocytes exposed in vivo and in vitro in preantral follicle culture to nocodazole Mutagenesis, January 1, 2005; 20(1): 65 - 75. [Abstract] [Full Text] [PDF]
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 H. M. Bolt and G. H. Degen Human Carcinogenic Risk Evaluation, Part II: Contributions of the EUROTOX Specialty Section for Carcinogenesis Toxicol. Sci., September 1, 2004; 81(1): 3 - 6. [Abstract] [Full Text] [PDF]
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C. Rosefort, E. Fauth, and H. Zankl Micronuclei induced by aneugens and clastogens in mononucleate and binucleate cells using the cytokinesis block assay Mutagenesis, July 1, 2004; 19(4): 277 - 284. [Abstract] [Full Text] [PDF]
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H. Norppa and G. C.-M. Falck What do human micronuclei contain? Mutagenesis, May 1, 2003; 18(3): 221 - 233. [Abstract] [Full Text] [PDF]
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