Topoisomerase II inhibition in mitosis produces numerical and structural chromosomal aberrations in human fibroblasts

Abstract

We investigated the effects of treatment of mitotic human fibroblasts with the topoisomerase II inhibitor etoposide (VP-16) on chromosome segregation at anaphase and the genetic consequence to daughter cells of topoisomerase inhibition during mitosis. The most striking effect of VP-16 treatment during mitosis was the production of anaphase cells with several entangled chromosomes (catenated anaphase cells). To analyze the effects of sister chromatid catenation at anaphase on the daughter cells, several interphase methodologies were applied to binucleated human fibroblasts that were blocked during cytokinesis. Post-treatment of mitotic cells with the cytokinesis inhibitor cytochalasin-B maintains the reciprocal products of a mitotic division in the same cytoplasm, allowing the distribution of whole chromosomes or chromosome fragments in daughter nuclei or micronuclei to be followed. The presence of micronuclei containing kinetochores, as detected by antikinetochore staining, suggested that VP-16 treatment during mitosis induces chromosome loss in binucleated fibroblasts. Induction of aneuploid cells for chromosomes 7 and 11 was observed by double in situ hybridization using chromosome-specific alphoid probes in binucleated fibroblasts. In addition, double in situ hybridization with adjacent alphoid and classical satellite DNA probes to chromosome 1 demonstrated that both numerical and structural aberrations contribute to the genetic effects of topoisomerase II inhibition in mitosis.