APPLICATION OF FLUORESCENCE IN-SITU HYBRIDIZATION TO STUDY THE RELATIONSHIP BETWEEN CYTOTOXICITY, CHROMOSOME-ABERRATIONS, AND CHANGES IN CHROMOSOME-NUMBER AFTER TREATMENT WITH THE TOPOISOMERASE-II INHIBITOR AMSACRINE

Amsacrine (4'-(9-acridinylamino)methanesulphon-m- anisidide) is an ant ileukemic drug which inhibits topoisomerase II (topo II) enzymes. We s tudied effects of two concentrations of amsacrine on the GM101 15A cel l line. This is a Chinese hamster line containing a single human chrom osome 4, which can be readily visualised using fluorescence in situ hy bridisation (FISH). The low amsacrine concentration slowed cell growth but did not cause significant arrest in the G2 phase of the cell cycl e, while a higher concentration caused more long-term effects on the g rowth of the cells and caused G2 arrest. Either concentration led to c hromosomal fragments which were lost with increasing time after treatm ent, and chromosomal translocations which appeared stable For at least 8 days after treatment. At the low concentration, the loss or gain of a single chromosome was a common event. The higher concentration led to polyploid cells, usually containing an uneven number of chromosome 4. We propose two mechanisms for aneuploidy by amsacrine (or related t opo II poisons), either of which can be readily detected using FISH. A t low drug concentrations, aneuploidy may occur directly through, for example, a failure to resolve catenated chromatids prior to anaphase. However, there has been considerable interest in the role of the tell division control (cdc) kinase and cyclins in regulating the mammalian cell cycle, and these may also be involved in the response of cells to high concentrations of topo II poisons. Cdc2 proteins and cyclins are involved in coordinating diverse activities during the M phase of the cell cycle, including catalysis of chromosome condensation and reorga nisation of microtubules to allow chromosome separation during mitosis . Chromosome damage by topo II poisons will lead to G2 arrest, which a llows the cells time to repair the damage. During this time, cyclin A and cdc2 levels will fall, preventing venting the cell from entering m itosis and effectively the clock to G1 and the ploidy to tetraploid. A neuploid cells will derive from polyploid cells through loss of extra chromosomes. (C) 1996 Wiley Liss, Inc.