Spindle poisons can induce polyploidy by mitotic slippage and micronucleate mononucleates in the cytokinesis-block assay

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1 Mutagenesb vol.13 no.2 pp , 1998 Spindle poisons can induce polyploidy by mitotic slippage and micronucleate mononucleates in the cytokinesisblock assay Azeddine Elhajouji 1, Monica Cunha and Micheline KirschVolders Laboratory for Human Genetics, Vrije Universiteit Brussel, Pleinlaan 2, 1050Brussels, Belgium The human in vitro cytokinesisblocked micronudeus (MN) assay has been extensively used for detection of clastogenic and aneugenic agents. In this test binucleate cells are generally considered to be the main target cell population for assessing genotoxic effect and almost no attention is paid to the biological information contained in mononucleate cells. In this study we analysed the frequencies of micronucleate mononucleates in a control population and after in vitro exposure to clastogens or aneugens. A clear increase in MN hi mononucleates was found only after exposure to aneugenic compounds. By means of fluorescence in situ hybridization using a chromosome 1specific probe we further analysed the proportion of mononucleate cells with and without MN which were tetrasomic (tetraploid) and would have been induced during aneugen treatment by mitotic slippage. The data indicate that treatment with nocodazole induces tetrasomy for chromosome 1 (tetraploidy) and an increase in MN frequency in mononucleate diploid and tetraploid lymphocytes. The results thus confirm that some mononucleates pass mitosis without chromatid segregation to daughter nuclei. These data suggest that MN in mononucleates may be useful to distinguish clastogens from aneugens and increase the sensitivity of the test Introduction The in vitro cytokinesisblocked micronucleus (MN) method has been shown to have many advantages. Essentially it discriminates between cells which did not divide from those which divided once or more and allows concurrent scoring of different endpoints, e.g. MN, chromosome loss, chromosome nondisjunction and apoptosis (for reviews see Fenech, 1997; KirschVolders et al, 1997). Therefore, this methodology has gained the favour of both research and genotoxicology laboratories: efforts are being made to introduce it into the test battery as a replacement for the in vitro chromosome aberration test and for assessment of aneuploidy induction (Aardema et al, 1998; Albertini and KirschVolders, 1997; Marzin, 1997). In the many studies performed with this method almost no attention has been paid to the biological information contained in mononucleate lymphocytes. It is commonly accepted that mononucleates represent a population of cells which did not divide in culture. This assumption is a simplified one, since mononucleate cells may also derive from cells which are insensitive to cytochalasin B (Surrales et al., 1994; Zijno et al., 1994) and/or cells which were blocked in metaphase after treatment with spindle poisons and progressed with or without separation of chromatids to the next cell cycle. Considering control of the separation of sister chromatids at the metaphase/anaphase transition, it was suggested by Hollo way et al. (1993) that some unknown protein involved in linkage of sister chromatids must be destroyed by the same machinery that proteolyses cyclin B. Recent data, moreover, indicate (Murray, 1995; Udvardy, 1996) that it probably takes place in three consecutive steps through protein ubiquitination. This implies that cyclin B destruction is required for chromosome decondensation, nuclear envelope reformation and cytokinesis, but not for chromatid separation. There is thus no reason to consider that cytochalasin B would interfere with chromatid separation in primary cells from normal individuals. In the presence of spindle poisons one may thus expect that some mononucleate cells in lymphocytes blocked by cytochalasin B may undergo chromatid separation, become polyploid (4N) and contain 4C or 8C DNA, depending on whether or not they are able to cross the Gl/S checkpoint of the next interphase. If no chromatid separation occurred the chromosomes should obviously show endoreduplication (2N, 4C or 8C). During recent years our laboratory has performed several studies with the cytochalasin B methodology comparing MN frequencies and characteristics in human lymphocytes exposed in vitro (Van Hummelen and KirschVolders, 1992; Elhajouji et al, 1994, 1995, 1997; KirschVolders et al, 1996; Tafazoli et al, 1995, 1996) or in vivo (Van Hummelen et al, 1993, 1994) to mutagens. From these data the following information is presented in this work: (i) background frequencies of MN in mononucleate and binucleate cells in a control population (nonexposed to known mutagens); (ii) frequencies of MN in mononucleate cells obtained after in vitro exposure to clastogens or aneugens; (iii) ploidy level of mononucleate cells after in vitro exposure to nocodazole. Materials and methods Chemicals Nocodazole (NOC; CAS ) was provided by Janssen Pharmaceutica (Beerse, Belgium). Carbendazim (MBC; CAS ) was purchased from Aldrich Chemie (Steinheim, Germany) and mebendazole (MEB; CAS ) from Sigma Chemical Co. (Brussels, Belgium). These three chemicals were dissolved in dimethylsulfoxide (DMSO; Merck, Darmstadt, Germany) for spectroscopy. Methyl methanesulfonate (MMS; CAS 66273) was purchased from Merck and dissolved in phosphatebuffered saline (PBS). Colchicine (COL; CAS 64868) and mitomycin C (MMC; CAS 50077) were purchased from Janssen Chemica (Beerse, Belgium) and dissolved in PBS. Cytochalasin B (Sigma Chemical Co.) was dissolved in DMSO and kept as a stock solution of 4 mg/ml at 20 C. Cultures Human peripheral blood samples were obtained from healthy volunteers not exposed to known mutagens. Blood was drawn by venipuncture and heparinized with Calparine 0 (Sanofi, Labaz, France). Lymphocytes were cultured in Ham's F10 medium supplemented with HEPES buffer (Gibco BRL, Bethesda, MD) containing 15% fetal calf serum (Gibco BRL) and 1% penicillin/ 'To whom correspondence should be addressed. Tel: ; Fax: ; aelhajou@vub.ac.be UK Environmental Mutagen Society/Oxford University Press

2 A.ElhnJo*Ajl, M.Cunha and M.KirschVoWers Background frequency of MNMononucleates Qrmph A B S S6 67 Range of MNMononndeatea (%o) Background frequency of bfnbinticleatea * 36 r 30 ^ 25 j Jill S6 67 Range of KQfMnucleatea (%o) Graph B Fig. 1. Percentage of individuals with a given spontaneous MN mononucleate frequency (A) and background frequencies of MN binucleates (B). All 240 healthy donors (230 men, 10 women) were included in this distribution. streptomycin (5000 IU/ml and 5000 ig/ml; Life Technologies, Paisley, UK) and incubated in 5% CO2 in a humidified incubator at 37 C. The lymphocytes were stimulated with 2% phytohaemaggluunin (PHA; Wellcome Diagnostics, UK) and treated with cytochalasin B (6 Hg/ml) at 44 h. After 72 h cultures were harvested. Cells were subjected to a cold hypotonic treatment (0.075 M KC1), immediately centrifuged and fixed three times with fixative (methanol:acetic acid, 3:1); an additional three drops of formaldehyde were included in the first fixation. The fixed cells were dropped onto slides using Pasteur pipettes, air dried and stored at 20 C. Treatment Chemicals were always added in a volume not exceeding 20 ul for isolated lymphocyte cultures. DMSO, if required, was always added at the same final concentration of 0.5%, including the DMSO used to dissolve cytochalasin B. The test products were added to the cultures 24 h after PHA stimulation and treatment lasted for 48 h, except with colchicine, where the treatment lasted for 19 h. Two cultures were prepared in parallel for each concentration. Fluorescence in situ hybridization (FISH) FISH with a probe for the centromeric region of chromosome 1 (puc1.77) was used. The probes were labelled by nick translation according to the instructions of the suppliers (Life Technologies). FISH was performed as described earlier (Elhajouji el ai, 1997). Slides were treated with RNase (Sigma) (0.1 mg/m] in 2X SSC) for 1 h and a mild pepsin solution (Sigma) (0.001% in 10 mm HC1) for 10 min in a 37 C water bath. The slides were denatured in 70% formamide, 2x SSC for 2 min and dehydrated in an ethanol series (50, 70 and 100%). The probes were denatured at 90 C and placed on slides. Following overnight hybridization at 37 C in a moist chamber the slides were washed with 50% formamide in 2X SSC at 42 C. Detection of the biotinylated, labelled probe for chromosome 1 was performed with avidin FTTC (fluorescein avidin D; Vector Laboratories, Burlingame, CA) and biotinylated goat antiavidin antibodies (Vector Laboratories), allowing signal amplification. After dehydration in an ethanol series (50, 70 and 100%) the cells were counterstained with ethidium bromide (Sigma) in /7phenylenediamine antifade solution. MN analysis Two cultures per concentration were analysed. One thousand mononucleate lymphocytes were examined per culture for the presence of one, two or more MN. All slides were coded and analysed with a Zeiss microscope at a magnification of 1250X. FISH analysis For FISH analysis at least 1000 mononucleate lymphocytes with and without MN per culture (two cultures per concentration) were examined for chromosome 1 distribution. The preparations were examined with a Zeiss Axioscop microscope (Carl Zeiss, Oberkochen, Germany) equipped with a filter (filter block 9, Zeiss) to visualize the fluoresceinlabelled probe and the orangered ethidium bromide counter staining. Statistics Statistical differences between controls and treated samples were determined with the x 2 test. Results Background frequencies of MN in mononucleate and binucleate cells in a control population (not exposed to known mutagens) Blood samples from 240 healthy donors (230 men and 10 women) were used to determine the background frequencies of MN in mononucleate cells. The classic in vitro cytokinesisblocked MN method was used. The frequency of micronucleate mononucleates (MN mononucleates) ranged from 0 to 5.60%o, with a median value of 0.99%c. The frequency distribution (Figure la) shows that 100% of analysed controls had between 0 and 5%o MN mononucleates and 50% had between 0 and l%o MN mononucleates. A parallel analysis was performed on micronucleate binucleates (MN binucleates) in the same population (Figure lb). The frequency distribution shows that 80% of the analysed controls had between 0 and 5 %o MN binucleates but only 9% had between 0 and l%o MN binucleates. 194

3 Induction of polyploidy by mitotlc slippage A Correlation coefficient: r n Graph A Age range (yean) Correlation coefficient? r Graph B i. _ " Age range (Tears) Fig. 2. A linear regression of the MN mononucleate (A) and MN binucleate (B) frequencies as a function of increasing male donor age. MN mononucleate and MN binucleate frequencies are plotted as means ± SE. As far as an age effect on the baseline frequency of MN mononucleates and MN binucleates in men is concerned, a statistically significant correlation was observed when different age clusters were constructed (Figure 2a and b). No comparison of spontaneous frequency of MN mononucleates and MN binucleates between men and women was performed since not enough women were available in this study. Frequencies of MN in mononucleate cells obtained after in vitro exposure to clastogens or aneugens. (MMS, MMC, colchicine, nocodazole, mebendazole and carbendazim) Basically, the MN mononucleates in the cytokinesisblocked assay on lymphocytes are cells that either did not undergo cell division (background level) or underwent an abnormal cell division which resulted in a MN mononucleate cell. The possibility of escaping from the cytokinesisblock might also contribute to the final frequency of MN mononucleates. As shown in Figure 3, all the tested aneugens induced a clear concentrationdependent increase in MN mononucleate frequencies, which became statistically significant at the highest concentrations. The concentrations that induced a statistically significant increase in MN mononucleates compared with those which induced a significant increase in MN binucleates (Elhajouji et ai, 1995) were higher for nocodazole, carbendazim and mebendazole and similar for colchicine. In contrast, the clastogens (MMS and MMC) did not induce an increase in frequencies of MN mononucleates (Figure 3)., Ploidy level of mononucleate cells To understand the mechanism of formation of MN in mononucleates we analysed the ploidy levels in mononucleate cells after in vitro exposure to nocodazole. It was assessed by means of FISH analysis with a chromosome 1specific (peri) centromeric probe. Figure 4 shows data from nocodazoletreated mononucleates in the cytokinesisblock assay. A clear increase in tetrasomic (tetraploid) mononucleate frequency was found with increasing concentration. The frequency of tetrasomic (tetraploid) MN mononucleates also showed a slight increase, which reached 41% of total MN mononucleates. It is clear from our data that in some mononucleates chromosomes indeed underwent condensation and chromatid segregation without separation into daughter nuclei. Discussion Several in vitro studies on human lymphocytes (Elhajouji et al., 1995, 1997; KirschVolders et al., 1996; Marshall et al., 1996; Zijno et al., 1996) showed that aneugens induce centromerepositive MN and chromosome nondisjunction and that a thresholdlike induction is associated with both mechanisms (Elhajouji et al, 1995, 1997). These two parameters assessing aneuploidy formation were analysed in binucleate human lymphocytes obtained by the cytokinesisblock methodology (Fenech and Morley, 1985; Fenech, 1997; KirschVolders et al, 1997). The main advantage of this method is that it allows discrimination between cells which divided once (binucleates) or more (trinucleates and terranucleates) and those which did not divide in culture. Recent findings in our laboratory (Cundari et al, 1998) demonstrated that human cell lines KS and K562, respectively expressing and not expressing p53, submitted to continuous exposure to nocodazole at a concentration which completely inhibits spindle formation can undergo mitosis without chromatid segregation but undergo Gl arrest by a p53 dependent mechanism. Indeed, in the absence of a functional mitotic spindle, maturation promoting factor (MPF) can be spontaneously inactivated, even when chromatid migration to the poles is hampered (a process known as mitotic slippage), giving rise to 4N, 4C cells (Andreassen and Margolis, 1994). These 4C polyploid cells either die after induction of apoptosis in the presence of p53 expression or cycle further, becoming 8C polyploid, in the absence of p53 expression. Considering these features, we expected that human lymphocytes from normal donors (expressing p53) would also escape mitotic arrest by mitotic slippage in the presence of nocodazole, giving rise with the cytokinesisblock method to tetraploid mononucleates with or without micronuclei. To test this hypothesis, we stepwise analysed: (i) spontaneous frequencies of MN in mononucleates versus binucleates from normal donors as a function of age in lymphocyte culture supplemented with cytochalasin B (data from preparations used in Van Hummelen et al, 1993, 1994, and from other unpublished work); (ii) frequencies of MN induced by aneugens versus clastogens (the same preparations as those used for the study of MN in binucleates; Elhajouji et al, 1995); (iii) frequencies of polysomy for chromosome 1 (and thus probably of polyploidy) in mononucleates obtained from human lymphocyte cultures exposed to nocodazole and submitted to the cytokinesisblock method. The answers to our questions are very clear. There is indeed an agedependent increase in mononucleates and binucleates 195

4 A.HhaJouJi, M.Cunha and M.KlrschVolders I MMS OOS Cone. 12 IS Fig. 3. Frequencies of MN mononucleates (%o MNmonoN) and MN binucleates (%* MNCB) as a function of increasing concentrations of the respective mutagens. with MN. This is not surprising for MN mononucleates since MN are formed in vivo in the donor before the in vitro cultivation period and represent spontaneous induction of MN; those found in binucleates show higher frequencies, probably because they correspond to MN induced both spontaneously in vivo and in vitro by the culture procedure. Similar increases in MN frequency with age have already been reported, especially in women >50 years (Catalan et ai, 1995), which in that case corresponds to preferential loss of the inactive X chromosome (Zijno et ai, 1996). Comparison of MN frequencies in mononucleates and binucleates after treatment with aneugens (nocodazole, mebendazole, colchicine and carbendazim) versus clastogens (MMS 196 and MMC) showed that aneugens, but not clastogens, clearly induced an increase in MN in mononucleates as well as in binucleates. In general, the frequencies of MN are higher in binucleates than in mononucleates, except for with colchicine. This suggests that the MN found in mononucleates obtained after 72 h in culture submitted to the cytochalasin B methodology essentially contain lost chromosomes and not acentric fragments characteristic of clastogenicity. The origin of these lagging chromosomes in mononucleates could be analysed by the FISH technology, which allows detection of chromosomal regions (e.g. centromeres or telomeres; Miller and Nusse, 1993; Elhajouji et ai, 1995) or chromosomespecific regions (e.g. the pericentromeric region

5 Induction of polyploidy by mitotic slippage Frequency ("&) 100 T mononucleates with 2 spots mcnonuclealct with 4 spots MNmommucleatti with 2 spots MNntoEonudcates with 4 spots Cooc. (ug/ml) Fig. 4. The frequencies of polysomy for chromosome 1 in raononucleates obtained from human lymphocyte cultures exposed in vitro to nocodazole. One thousand mononucleate lymphocytes per culture with and without MN were examined for chromosome 1 polysomy. All treatments were performed in replicate. of chromosome 1; Eastmond and Rupa, 1995; Marshall et al, 1996; Elhajouji et al, 1997). With the same methodology we studied the proportion of mononucleate cells with and without MN which were tetrasomic and would have been induced during aneugen treatment by mitotic slippage. The data indicate, on the one hand, that treatment with nocodazole induces tetraploidy (as measured by tetrasomy for chromosome 1) and, on the other hand, that aneugens induce an increase in MN frequencies in mononucleate diploid and tetraploid lymphocytes. These increases are accompanied by a relative decrease in binucleate cells (and trinucleate/tetranucleate cells). The results thus confirm that some mononucleates pass mitosis without chromatid segregation to daughter nuclei. Whether the MN found in the tetraploid mononucleates result from chromosome 'lagging' during mitotic slippage occurring in the absence of a spindle is unclear. The increase in MN in the diploid mononucleates is more questionable and does not exclude the fact that some of the cells treated with nocodazole completed division before cytochalasin B addition and failed to divide further. Another possibility is that the treated cells underwent a normal cell division, being insensitive to cytochalasin B, giving rise to two mononucleate cells instead of the expected binucleate. The relative frequencies of mononucleates and binucleates with and without MN do not allow us to give a definitive answer, since the increase in mononucleates after exposure to a mutagen may also result from cell cycle delay. What are the implications of these findings? From a practical point of view related to use of the in vitro MN test to detect clastogens/aneugens, it indicates that scoring of MN in mononucleates may be an additional interesting endpoint to distinguish clastogens from aneugens and to increase the sensitivity of the test, taking into account the timing of 0.04 treatment, addition of cytochalasin B and sampling. The MN increase, however, does not appear earlier in mononucleates than in binucleates and thus cannot be used as an earlier marker. Nondisjunction remains the earliest marker for aneuploidy induced by spindle inhibitors (Elhajouji etal., 1997). Analysis of MN in both mononucleate and binucleate cells on the same slide is also important for determination of MN kinetics in biomonitoring studies (Channarayappa et al, 1992); indeed, discrimination of newly formed MN (MN in binucleates) and those that have persisted from any initial chemical effect (MN in mononucleates) is possible. From a more fundamental point of view, it indicates that the mitotic slippage described by our laboratory in human cell lines exposed in vitro to nocodazole can be extended to primary human lymphocytes exposed to spindle inhibitors. Treatment with aneugens in vitro or with aneugenic chemotherapeutic drugs in vivo will therefore induce polyploidization in p53 deficient cells or tumours respectively. When testing is performed on p53deficient cell lines the results may be misleading as compared with primary p53 expressing cells. When therapy is applied under these conditions the appearance of secondary tumours is favoured and highly probable. Acknowledgements We would like to thank C.Durang for her technical assistance. This work was supported by the Belgian Federal Office for Scientific, Technical and Cultural Affairs (OSTC) and by the CEC, contract no References AardemaXJ., Albertini.S., Ami,R, HendersonXM., KirschVolders,M., MackayJJW., Sarrif^AJVl., StringerJ)JV. and Taalma^R.D.F. (1998; Aneuploidy: a report of an ECETOC task force. Muuu. Res., in press Albertini,S. and KirschVblders,M. (1997) Summary and conclusions on the MNT in vitro and implication on testing strategies. Mutat Res., 392,

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