Micronuclei in neonatal lymphocytes treated with the topoisomerase II inhibitors amsacrine and etoposide

Mutation Research, 319 (1993) 215-222

215

© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1218/93/$06.00

MUTGEN 01930

Micronuclei in neonatal lymphocytes treated with the topoisomerase II inhibitors amsacrine and etoposide Anne Slavotinek, P.E. Perry and A.T. Sumner MRC Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, UK

(Received 27 January 1993) (Revision received 4 May 1993) (Accepted 11 May 1993)

Keywords: Micronuclei; CREST staining; Topoisomerase II inhibitors; Non-disjunction; Neonatal lymphocytes; Lymphocytes,

neonatal; Amsacrine; Etoposide

Summary It has been suggested that the enzyme topoisomerase II may be important in chromosome segregation due to the role played by the enzyme in decatenating the intertwined D N A molecules that result from D N A replication. Inhibition of the enzyme has been found by some workers to inhibit chromatid separation in mammalian cells, while others have reported that the passage of cells through mitosis is unaffected. Inhibition of the enzyme with topoisomerase II inhibiting drugs also results in the formation of micronuclei as a consequence of D N A damage. We have used the micronucleus assay with C R E S T staining to investigate whether the micronuclei formed in neonatal lymphocytes after inhibition of topoisomerase II are formed from whole chromosomes, implying non-disjunction, or acentric fragments. We found that treatment with both amsacrine and etoposide caused a dose-related increase in the n u m b e r of C R E S T negative micronuclei, with only a very small increase in the number of C R E S T positive micronuclei at high concentrations of the compounds. Although we cannot conclude from our experiments that treatment with topoisomerase II inhibitors does not affect the segregation of neonatal lymphocytes, the production of C R E S T negative micronuclei suggests that segregation abnormalities are less important than other mechanisms which may cause cytotoxicity from exposure to these compounds.

The nuclear enzymes topoisomerase I and II resolve the topological and conformational p r o b lems that occur in D N A processes such as replication and transcription (for reviews, see D ' A r p a and Liu, 1989; Lui, 1989). Type I topoisomerases Correspondence: Dr. Anne Slavotinek, MRC Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, UK. Fax 31-343-2620.

form transient, enzyme-bridged breaks in a single strand of DNA, and catalyse the relaxation of DNA. Type II topoisomerases introduce a similar but double-stranded break into D N A through which another double-strand may be passed, allowing the enzyme to unknot, decatenate or relax supercoiled DNA. It has been suggested that topoisomerase II might play a role in chromosome segregation. Newly replicated D N A molecules are inter-

216

twined, and require decatenation prior to chromosome separation (Hsieh and Brutlag, 1980; Sundin and Varshavsky, 1981). Research involving yeasts has shown that DNA topoisomerase II is required at the time of mitosis (Holm et al., 1985), and that the enzyme is also necessary for chromosome segregation (di Nardo et al., 1984; Uemura and Yanagida, 1986; Holm et al., 1989). Topoisomerase II is necessary for the segregation of daughter molecules during simian virus DNA (SV40) replication, although both topoisomerases can unlink DNA for fork propagation (Yang et al., 1987). A study using mammalian cells has found that topoisomerase II activity was necessary for chromatid separation (Downes et al., 1991), although other workers have reported that inhibition of topoisomerase II does not block the passage of the cells through mitosis (Rowley and Kort, 1989; Sumner, 1992). Sumner's experiments did not exclude the possibility that segregation was impaired, as the cytological methods used may have failed to detect lagging chromosomes due to the small number of anaphase cells or insufficient separation of daughter groups of chromosomes in a particular preparation. In recent years, micronuclei have increasingly been used to assess chromosome damage (Heddle et al., 1991). They can arise from a variety of mechanisms that include the failure of damaged cells to incorporate acentric fragments or whole chromosomes into daughter nuclei at cell division. Micronuclei containing whole chromosomes, arising as a result of non-disjunction, can be distinguished from fragments without centromeres by staining the kinetochores of the intact chromosomes with anti-kinetochore antibody from CREST serum (Moroi et al., 1980). The detection of CREST positive micronuclei has thus been used to identify agents that induce aneuploidy (Vig and Swearngin, 1986; Thomson and Perry, 1988; Eastmond and Tucker, 1989; Fenech and Morley, 1989). Topoisomerase II inhibitors stabilise the DNA-topoisomerase II complex during strand passage (the reaction intermediate so formed is termed the cleavable complex) and prevent the rejoining of DNA breaks (Lui, 1989). The DNA isolated from cells after drug treatment contains topoisomerase II subunits attached with covalent

bonds, and strand breaks are detectable when the protein is degraded. Treatment with topoisomerase II inhibitors has been shown to result in extensive chromosome damage (Huang et al., 1973; Rowley and Kort, 1989; Sumner, 1992). It is therefore not surprising that treatment with topoisomerase II inhibitors has been shown to cause micronuclei, and that this finding has been reported in mammalian cells (Wilson et al., 1984; Lock and Ross, 1990b; Holmstrom and Winters, 1992). However, the aforementioned research did not use anti-kinetochore antibody staining, and thus did not utilise the assay to investigate the effect of topoisomerase II inhibition on cell segregation. We have used the micronucleus test with CREST staining to determine whether the micronuclei formed after inhibition of topoisomerase II result from whole chromosomes or acentric fragments. Materials and methods

The culture medium used in the following experiments consisted of RPMI 1640 (Gibco) supplemented with 10% fetal calf serum, 2 mM glutamine, 100 I.U. penicillin per ml, 100 I.U. streptomycin per ml, 0.5% NaHCO 3 and 1% phenol red. A supplementary antibiotic solution was added to the culture medium as the blood samples could not always be obtained under sterile conditions. The solution consisted of tylosine at 40 /xg/ml (Life Technologies, Detroit), Kanamycin at 5 / x g / m l (Sigma, St. Louis), Gentamycin at 8 /xg/ml (Sigma, St. Louis) and Trobicin at 4 /xg/ml (Vestric, Edinburgh). Preliminary experiments showed that these antibiotics did not influence the spontaneous micronucleus frequency of neonatal lymphocytes. RPMI 1640 supplemented with 2% fetal calf serum was used as a wash medium. Lymphocytes were separated from neonatal cord blood samples (Lymphoprep, Nycomed) and washed once. Contaminating red blood ceils were lysed by the addition of 5-10 ml of ice-cold lysis buffer (155 mM ammonium chloride, 10 mM potassium hydrogen carbonate and 0.1 mM diaminoethanetetra-acetic acid (EDTA) in double distilled water) to the cells for up to 10 min. After a second wash, 4 to 8 2.5-mi aliquots containing

217 2.5 × 10 6 lymphocytes were dispensed to 10 ml tissue culture tubes for each experiment. Reconstituted phytohaemagglutinin HA15 (Wellcome Diagnostics, UK) and the supplementary antibiotic solution were added to the culture tubes to a final concentration of 1%. The tubes were incubated in a humidified incubator containing 5% CO2 at 37°C. 4'-(9-Acridinylamino)methanesulphon-m-anisidine (amsacrine; 5 mM in dimethyl sulphoxide (DMSO); W a r n e r - L a m b e r t / P a r k e Davis) and etoposide (VP-16; 5 mM in DMSO; BristolMeyers Squibb) were both stored as stock solutions at -20°C. The compounds were thawed and diluted with culture medium immediately prior to use. Two types of experiment were performed. In the first group of experiments, the topoisomerase II inhibitors were added to the culture tubes after 41 h, and were present throughout the remainder of the incubation period. In the second group of experiments, the topoisomerase II inhibitors were added after 43.5 h for 30 min only. The compounds were removed by washing the cells twice with prewarmed wash medium, and the lymphocytes resuspended in fresh culture medium, phytohaemagglutinin and the supplementary antibiotic solution. Cytochalasin B (Sigma, St. Louis) at a final concentration of 3 /~g/ml was added to all cultures in both groups of experiments after 44 h. The cytochalasin B was stored as a stock solution in DMSO at a concentration of 2 m g / m l at 20oc. After a total of 72 h incubation, the lymphocytes from both groups of experiments were washed once with wash medium and resuspended for 5 min in 1-2 ml of hypotonic solution consisting of culture medium, double distilled water and DMSO in a ratio of 1 : 4 : 0.02 (v : v). Cytocentrifuge preparations were made by spinning at 1200 rpm (Cytospin, Shandon) for 5 min onto acid-alcohol cleaned slides. The slides were allowed to air dry prior to fixation in 90% methanol at - 2 0 ° C for 10 min. The slides were stained with C R E S T antibody essentially according to the method described by Thomson and Perry (1988). Staining of the cell nuclei and cytoplasm was achieved with a combi-

nation of 4',6-diamidino-2-phenylindole (DAPI; final concentration 0.5 /xg/ml; Sigma, St. Louis) and propidium iodide (final concentration 6 /xg/ml; Sigma, St. Louis) added to mountant containing 1 : 1 glycerol and AFT10, an antifadent (Citifluor). Slides were scored with an Ortholux II fluorescent microscope (Leitz, Germany) under 100 x objective magnification. The DAPI stain produced bright blue fluorescence of the DNA in cell nuclei and micronuclei, and was viewed using filter block A. The propidium iodide produced weak red fuorescence of the cytoplasm and strong fluorescence of the nucleus under filter block N2, and was used to identify binucleate cells. The CREST antibody was seen as green fluorescence of the chromosome kinetochores using filter block I3. The number of nuclei in 500 consecutive cells was scored for each slide. The number of micronuclei in 500 binucleate cells was then counted for each slide, and each micronucleus was examined for C R E S T staining. In the experiments involving a short incubation with the topoisomerase II inhibitors, a reduced frequency of binucleate cells at the higher drug concentrations meant that fewer cells were available for scoring micronucleus frequency. 250 binucleate cells were scored for micronuclei on the slides resulting from 1.0 p.M and 2.0/xM amsacrine and 8.0 tzM and 16.0 ~ M etoposide. At 4.0 IzM amsacrine and 32.0 IzM etoposide, 75-100 binucleate cells were scored. The criteria used for the scoring of micronuclei were based on the recommendations made at the Health and Safety Workshop on micronuclei (Arlett et al., 1989). (1) The cells scored for micronuclei must be clearly seen as binucleate. A cell containing two nuclei without complete separation of the daughter nuclei was considered acceptable. (2) A micronucleus must be less than a fifth of the size of the smaller daughter nucleus, and sufficiently separated from the main nuclei to ensure that nuclear extrusions would not be counted. (3) The micronucleus must stain with DAPI, but could do so at a different intensity from that of the daughter nuclei. (4) The number of micronuclei in each binucleate cell must be scored.

218 E a c h slide was assessed for a d e q u a t e C R E S T staining before scoring. Every m i c r o n u c l e u s was e x a m i n e d for C R E S T staining in several focal p l a n e s to avoid missing positive m i c r o n u c l e i d u e to different focal depths of the cells o n the slides. Nevertheless, occasional failure to detect a bright, F I T C - p o s i t i v e body in a small m i c r o n u c l e u s cannot be discounted. All e x p e r i m e n t s were p e r f o r m e d a m i n i m u m of 3 times. Results

I n the first group of experiments, c o n t i n u o u s exposure of the lymphocytes to a m s a c r i n e or etoposide for 31 h p r o d u c e d a decrease in the p r o p o r t i o n of b i n u c l e a t e cells (Table 1), b u t a n increase in the fraction of b i n u c l e a t e cells cont a i n i n g m i c r o n u c l e i ( T a b l e 1 a n d Fig. 1). T h e r e was no significant increase in the n u m b e r of C R E S T positive micronuclei. I n the second series of experiments, the lymphocytes were exposed to higher c o n c e n t r a t i o n s of the t o p o i s o m e r a s e II inhibitors for 30 m i n

~,

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THE NUMBER OF BINUCLEATE (BN) CELLS AND MICRONUCLEI (MN) AFTER THE TREATMENT OF NEONATAL LYMPHOCYTES FOR 31 h WITH AMSACRINE AND ETOPOSIDE All values are the mean from 3 independent Expts. Drug concentration (/zM)

% ofbinucleate cells in 500 cells

% of cells MN/BN with MN cell in 500 BN cells

CREST positive MN/BN cell

Amsacrine 0 0.025 0.05 0.1

63.9 52.8 49.4 34.1

0.67 1.9 3.3 5.6

0.007 0.02 0.034 0.058

0.003 0.003 0.003 0.004

Etoposide 0 0.05 0.1 0.2

64.9 55.5 51.1 25.5

0.6 2.1 3.9 6.3

0.006 0.021 0.041 0.066

0.004 0.004 0.005 0.005

before i n c u b a t i o n in drug-free m e d i u m . T h e s e e x p e r i m e n t s also p r o d u c e d a decrease in the perc e n t a g e of b i n u c l e a t e cells (Table 2), a n d a dose

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Fig. 1. The frequency of micronuclei per binucleate cell and CREST positive micronuclei per binucleate cell after treatment of neonatal lymphocytes with increasing concentrations of amsacrine and etoposide for 31 h. The dark symbols indicate values from independent experiments, the light symbols indicate the mean values for 3 Expts. Symbols from similar values are superimposed upon each other.

219 dependent increase in the fraction of binucleate cells containing micronuclei (Table 2 and Fig. 2). At the higher concentrations of amsacrine and etoposide there was a modest increase in the number of C R E S T positive micronuclei, although the majority of micronuclei remained C R E S T negative (Fig. 2). Discussion

In the experiments described in this paper, we have tested the hypothesis that inhibition of topoisomerase II activity might disturb chromosome segregation, causing non-disjunction and an increase in the n u m b e r of C R E S T positive micronuclei that could be seen after cell division. We chose to use neonatal lymphocytes for these experiments as they have a low baseline micronucleus frequency and a low incidence of spontaneous non-disjunction when compared to lymphocytes from adult donors (Ogadiri et al., 1990). We initially chose to add the topoisomerase II inhibitors for the entire time that the lymphocytes were cultured with cytochalasin B to avoid possible recovery of impaired enzyme activity on removal of the drugs (Pommier et al., 1988). With

this approach, we were unable to reach the concentrations of the compound used by previous researchers to assess the effects of enzyme inhibition on mammalian cell segregation (Downes et al., 1991). Further increases in the drug concentrations caused excess cell death, which made scoring the slides difficult due to increasing artefact. Continued presence of the drugs may also have led to cell damage and death due to mechanisms other than topoisomerase II inhibition. In our experiments, prolonged incubation with amsacrine at 0.1 /~M and etoposide at 0.2 /~M resulted in a greater frequency of binucleate cells containing micronuclei than that obtained with the same concentrations of drug for a shorter incubation time. In the second group of experiments, a shorter exposure of the lymphocytes to the topoisomerase II inhibitors prior to incubation in drugfree medium permitted the effects of higher concentrations of the drugs to be assessed. Our experiments using etoposide were able to reach the concentrations at which abnormalities of segregation have sometimes been seen (Downes et al., 1991). Although there was a modest increase in 0.550

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Fig. 2. The frequency of micronuclei per binucleate cell and CREST positive micronuclei per binucleate cell after treatment of neonatal lymphocyteswith increasing concentrations of amsacrine and etoposide for 0.5 h. The dark symbols indicate values from independent experiments. The light symbolsindicate the mean values from 3 Expts.

220 TABLE 2 THE NUMBER OF BINUCLEATE(BN) CELLS AND MICRONUCLEI (MN) AFTER THE TREATMENT OF NEONATAL LYMPHOCYTES FOR 0.5 h WITH AMSACRINE AND ETOPOSIDE All values are the mean from 3 independent Expts. The numbers in brackets after the values for the percentage of cells containing micronuclei indicate the number of binucleate cells scored. Drug concentration (p~M)

% of binucleate cells in 500 cells

% of cells withMN in 75-500 BN cells

MN/BN cell

CREST positive MN/BN cell

60.7 51.0 44.2 28.5 17.8 4.1

0.47 (500) 1.0 (500) 5.5 (500) 18.7 (250) 34.4 (250) 49.3 (100)

0.005 0.011 0.063 0.233 0.476 0.813

0.002 0.003 0.005 0.015 0.017 0.037

64.8 57.6 58.1 50.1 34.6 18.9 6.3 3.5

0.27 (500) 1.1 (500) 2.8 (500) 5.2 (500) 9.7 (500) 18.5 (250) 26.7 (250) 27.8 (75-100)

0.003 0.011 0.029 0.057 0.113 0.253 0.41 0.407

0.001 0 0.001 0.003 0.005 0.007 0.03 0.054

Amsacrine

0 0.1 0.5 1.0 2.0 4.0 Etoposide

0 0.2 1.0 2.0 4.0 8.0 16.0 32.0

the number of C R E S T positive micronuclei at the highest drug concentrations, the majority remained C R E S T negative. Flow cytometry performed at 43.5 h on 5 independent cultures to examine the cell-cycle distribution of the lymphocytes at the time of drug treatment showed that the majority of lymphocytes were in the S phase of the cell cycle (42.2-66.5%), with a small number (2.1-8.8%; mean = 5.5%) in the G 2 / M phase of the cell cycle (data not shown). Treatment in both of the above cell-cycle stages is likely to have resulted in DNA-strand breaks as the result of cleavable complex formation (Estey et al., 1987). The cells in the G 2 / M phase of the cell cycle at the time of drug treatment may have entered or continued mitosis in the presence of the drugs, and the resultant damage would be detected as C R E S T negative micronuclei in our experiments. However, the DNA-strand breaks resulting from cleavable complex formation are

rapidly reversed when cells are transferred to drug-flee medium (Pommier et al., 1984; Long et al., 1985; Kohn et al., 1987). Cleavable complex formation has also been dissociated from the cell killing effects of the drug (Bertrand et al., 1990; Chatterjee et al., 1990). An alternative explanation for the C R E S T negative micronuclei is suggested by the finding that etoposide has been shown to induce endonucleolytic cleavage without the continued presence of the drug (Kaufmann, 1989). It is possible that the D N A damage so formed is detectable at cell division as C R E S T negative micronuclei in our experiments. It was in the group of G 2 / M cells, however, that we hoped to see an increase in the number of C R E S T positive micronuclei that would have indicated impaired segregation due to enzyme inhibition at the higher concentrations of the drugs. Although the higher concentrations of the drugs did result in a small rise in the number of C R E S T positive micronuclei, perhaps indicating that faulty segregation could occur infrequently, firm conclusions are difficult to draw from our experiments. Dose-related increases in the frequency of kinetochore-positive micronuclei have also been observed when testing high concentrations of agents which are thought principally to be clastogenic (Eastmond and Tucker, 1989). Downes et al. (1991) found that the concentrations of etoposide required to inhibit segregation were much higher than those required to induce G2 block in Muntjac cells. They also found that ultimately lethal concentrations of topoisomerase II inhibitors were required for extensive inhibition of segregation. Our results indicate that similar concentrations may be needed for neonatal lymphocytes, and suggest that abnormalities of segregation play a minor role in the cytotoxic mechanisms of the topoisomerase II inhibitors. Treatment in the phases of the cell cycle before late G2 is likely to have elicited G2 arrest as a result of the drug-induced D N A damage altering the cellular processes regulating the progression of cells from G2 to mitosis (Tobey, 1975). In the case of etoposide, inactivation of p34 cdc2 kinase activity has been causally related to G2 arrest (Lock and Ross, 1990a,b). Arrest in the G2 phase of the cell cycle has been shown to occur with treatment by many topoisomerase II in-

221

hibitors (Kalwinsky et al., 1983; Del Bino et al., 1991), and it is likely that G2 arrest is responsible for most of the decrease in binucleate cells in our experiments. However, treatment with topoisomerase II inhibitors has also been shown to inhibit S phase cycle traverse, which could also have contributed to the increase in mononucleate cells in our experiments (Krishan et al., 1975). In sum, we did not observe a significant increase in C R E S T positive micronuclei that could unequivocally be attributed to non-disjunction after the treatment of neonatal lymphocytes with amsacrine and etoposide. Although our experiments do not exclude a role for topoisomerase II in chromosome segregation (decatenation of intertwined D N A molecules could occur prior to the entry of cells into mitosis), the high concentrations required to demonstrate a small increase in C R E S T positive micronuclei and the formation of many C R E S T negative micronuclei at these concentrations suggest that the inhibition of segregation does not play a major part in the cytotoxic effects caused by these compounds.

Acknowledgements We wish to thank the midwifery staff at the Simpson Memorial Maternity Pavilion, Edinburgh, for collection of neonatal blood samples and W a r n e r - L a m b e r t / P a r k e - D a v i s and BristolMyers Squibb for gifts of amsacrine and etoposide, respectively. The authors are also grateful to Eric Miller for his help with the flow cytometry analysis. Anne Slavotinek is especially grateful to the National Radiological Protection Board for funding her work.

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