Fragmentation of centromeric DNA and prevention of homologous chromosome separation in male mouse meiosis in vivo by the topoisomerase II inhibitor etoposide

Marko Kallio 0 Jaana Lahdetie 0 0 Department of Medical Genetics, University of Turku , Kiinamyllynkatu 10, Turku 20520-FIN, Finland 'To whom correspondence should be addressed - The mechanism of action of the topoisomerase II inhibitor etoposide (VP-16) was investigated in male mouse meiosis using the spermatid micronucleus (MN) test and two molecular cytogenetic approaches: (i) fluorescence in situ hybridization (FISH) with a mouse centromere specific minor satellite DNA probe,* and (ii) immunolabeUing of kinetochore proteins with CREST autoimmune serum. VP16 caused significant increases in the frequencies of MN at all meiotic stages studied. VP-16 induced MN showed significantly elevated frequencies of centromeric hybridization signals compared to the controls. Similarly, after CREST immunostaining the majority of MN induced by the drug showed kinetochore signals when meiotic S phase and diplotene-diakinesis were treated. This would suggest that most induced MN were due to lagging of whole chromosomes. However, more than 80% of the small MN observed were signal-positive and a large pool of minute MN almost exclusively (92%) contained a kinetochore or centromere-DNA signal. This indicates that VP-16 causes chromosome fragmentation at centromeres. In addition, arrested first division (MI) anaphase figures with stretched bivalent(s) at the spindle equator were observed when diplotene-diakinesis and MI were targeted. Moreover, many small and medium size MN had two centromere or kinetochore signals at opposite sides, suggesting that inhibition of topo II at MI causes lagging of whole bivalents. Together, these results indicate that VP-16 acts by several genotoxic mechanisms at male meiosis: (i) fragmentation of centromeres possibly as a result of inhibition of the DNA strand religation reaction in a topoisomerase II mediated decatenation process of sister centromeres; and (ii) the induction of aneuploidy as a result of failures in separation of homologous chromosome arms possibly due to disturbances of chiasma resolution and decatenation processes during MI. Our results indirectly suggest that topoisomerase II plays an important role in male meiosis and its activity is needed at the metaphase-anaphase transition of both meiotic divisions for proper chromosome disjunction. Introduction DNA topoisomerase II (topo II) is an essential enzyme for DNA integrity due to its ability to untangle sister DNA strands that are topologically linked after DNA replication. In mammalian cells, there are two types of topo II that are differentially expressed and regulated (Drake et al, 1989a). Topo Da, a 170 kDa form, is found in proliferating cells and is regulated during the cell cycle (Heck et al, 1988; Woessner et al, 1991), while the 180 kDa (i-form is less regulated and is found in both proliferating and quiescent cells (Woessner et al, 1991). Experiments with yeast (Holm et al, 1985, 1989; Uemura et al, 1987), frog egg extract (Shamu and Murray, 1992) and mammalian cell studies in vitro (Downes et al, 1991) all show that topo II is required at the time of sister chromatid segregation. Moreover, Rose et al (1990) and Rose and Holm (1993) have suggested a role for topo II in the resolution of recombined homologous chromosomes and in resolving tangles between nonhomologous chromosomes during meiosis I of yeast. In addition, topo II activity is also required for chromosome condensation (Uemura et al, 1987; Adachi, 1991). Topo n, especially in its a-form, is thought to be an important structural component of the mitotic chromosomal scaffold (Earnshaw et al, 1985) and is associated with the chromatin and synaptonemal complex of pachytene and diplotene chromosomes of male chickens (Moens and Earnshaw, 1989). Whether the localization of topo II in mitotic and meiotic chromosomes denotes strictly its structural role (Gasser and Laemmli, 1987) or not (Hirano and Mitchison, 1993) or is more an indication of requirement for the enzyme during chromosomal condensation and segregation remains to be established. The use of inhibition of topo II plays a major role in recent development of cancer chemotherapy. Many potent clinical drugs in use such as epipodophyllotoxins (VP-16 and VM26), anthracyclines (doxorubicin and daunorubicin), acridines (m-amsacrine) and anthracenediones (mitoxantrone) target topo II by stabilizing the enzyme-mediated DNA cleavage complex and, thus, inducing a covalent complex of topo II and DNA, which blocks DNA religation (Chen et al, 1984; Robinson and Osheroff, 1991). Recently, many new topo H-directed agents such as fo(2,6-dioxopiperazine) derivates (ICRF-159, ICRF-187 and ICRF-193) and the barbiturate derivative Merbarone have been demonstrated to inhibit normal chromosome segregation (Clarke et al, 1993; Gorbsky, 1994; Chen and Beck, 1995) by inhibiting topo II without stabilizing topo IIDNA covalent complexes (Drake et al, 1989b; Tanabe et al, 1991), but rather by affecting some unidentified catalytic step in a topo H-mediated reaction. This demonstrates that there are different mechanisms of inhibition of topo II function in target tissues which are not fully understood. Etoposide (VP-16) is one of the most studied anticancer drugs with widespread clinical use (Henwood and Brogden, 1990). It has improved the treatment of germ cell tumours, small-cell and non-small-cell lung carcinomas, and acute lymphocytic leukaemia. However, secondary leukaemias have been reported in patients treated with etoposide-containing therapy (Pui et al, 1991; Nichols et al, 1993; Winick et al, 1993). The molecular mechanism of action of VP-16 in vitro and the stereo-configuration of the cleavable complex is only partially known (reviewed in Anderson and Berger, 1994). Moreover, recent data obtained from different groups show some discrepancy in the mechanism of action of the drug in vivo; VP-16 has been shown to induce chromosomal fragmentation in mammalian cells in vitro (Sumner, 1992) and in vivo (Agerwal et al., 1994), while induction of aneuploidy is suggested by germ cell studies (Kallio and Lahdetie, 1993; Mailhes et al., 1994). We addressed the question, are there different mechanisms of action or different cellular targets in meiotic systems compared with mitotic cells? We used two molecular cytogenetic approaches, fluorescence in situ hybridization (FISH) with a mouse minor satellite DNA probe and immunofluorescent labelling of the kinetochores with calcinosis-Raynaud's phenomenon-oesophageal dismobility-sclerodactyly-telangiectasia syndrome of scleroderma (CREST) autoimmune serum, to investigate whether the formation of meiotic micronuclei (MN) during divisions after VP16 treatment is due to non-disjunction and lagging of a whole chromosome(s) with assembled kinetochores or is an indication of breakage of pericentromeric DNA of meiotic chromosomes of the male mouse. In our previous work (Kallio and Lahdetie, 1993), where the mouse major satellite DNA probe was utilized, (...truncated)