Kallio M, Lähdetie J
Department of Medical Genetics, University of Turku, Finland.
Mutagenesis. 1996 Sep;11(5):435-43. doi: 10.1093/mutage/11.5.435.
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) immunolabelling of kinetochore proteins with CREST autoimmune serum. VP-16 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.
利用精子细胞微核（MN）试验以及两种分子细胞遗传学方法，对拓扑异构酶II抑制剂依托泊苷（VP - 16）在雄性小鼠减数分裂中的作用机制进行了研究：（i）使用小鼠着丝粒特异性小卫星DNA探针进行荧光原位杂交（FISH）；（ii）用CREST自身免疫血清对动粒蛋白进行免疫标记。在所有研究的减数分裂阶段，VP - 16均导致MN频率显著增加。与对照组相比，VP - 16诱导产生的MN显示着丝粒杂交信号频率显著升高。同样，在CREST免疫染色后，当减数分裂S期和双线期 - 终变期受到处理时，该药物诱导产生的大多数MN显示出动粒信号。这表明大多数诱导产生的MN是由于整条染色体滞后所致。然而，观察到的超过80%的小MN呈信号阳性，并且大量微小MN几乎完全（92%）含有动粒或着丝粒 - DNA信号。这表明VP - 16会导致着丝粒处的染色体断裂。此外，当双线期 - 终变期和减数第一次分裂（MI）作为靶点时，观察到在纺锤体赤道处有拉长的二价体的停滞的第一次分裂（MI）后期图像。而且，许多中小尺寸的MN在相对两侧有两个着丝粒或动粒信号，这表明在MI期对拓扑异构酶II的抑制会导致整条二价体滞后。总之，这些结果表明VP - 16在雄性减数分裂中通过多种遗传毒性机制发挥作用：（i）着丝粒断裂可能是由于在拓扑异构酶II介导的姐妹着丝粒解连环过程中DNA链再连接反应受到抑制；（ii）由于同源染色体臂分离失败可能导致非整倍体的诱导，这可能是由于MI期交叉点分辨率和解连环过程受到干扰所致。我们的结果间接表明拓扑异构酶II在雄性减数分裂中起重要作用，并且在减数分裂两个阶段的中期 - 后期转换过程中其活性对于正确的染色体分离是必需的。
