Application of fluorescence in situ hybridisation 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 antileukemic drug which inhibits topoisomerase II (topo II) enzymes. We studied effects of two concentrations of amsacrine on the GM10115A cell line. This is a Chinese hamster line containing a single human chromosome 4, which can be readily visualised using fluorescence in situ hybridisation (FISH). The low amsacrine concentration slowed cell growth but did not cause significant arrest in the G2 phase of the cell cycle, while a higher concentration caused more long-term effects on the growth of the cells and caused G2 arrest. Either concentration led to chromosomal fragments which were lost with increasing time after treatment, 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 topo II poisons), either of which can be readily detected using FISH. At 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 cell 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 reorganisation of microtubules to allow chromosome separation during mitosis. Chromosome damage by topo II poisons will lead to G2 arrest, which allows the cells time to repair the damage. During this time, cyclin A and cdc2 levels will fall, preventing the cell from entering mitosis and effectively resetting the clock to G1 and the ploidy to tetraploid. Aneuploid cells will derive from polyploid cells through loss of extra chromosomes.
Ferguson
L R
LR
Cancer Research Laboratory, University of Auckland Medical School, New Zealand.
Whiteside
G
G
Holdaway
K M
KM
Baguley
B C
BC
D000677:Amsacrine / Q000633:toxicity*; D000704:Analysis of Variance; D000782:Aneuploidy; D000818:Animals; D000970:Antineoplastic Agents / Q000633:toxicity*; D016466:CHO Cells; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002869:Chromosome Aberrations / Q000139:chemically induced / Q000235:genetics*; D025063:Chromosome Disorders; D002894:Chromosomes, Human, Pair 4 / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D016195:G2 Phase / Q000187:drug effects; D006801:Humans; D006822:Hybrid Cells; D017404:In Situ Hybridization, Fluorescence; D008677:Metaphase / Q000187:drug effects; D011123:Polyploidy; D059005:Topoisomerase II Inhibitors
D000970:Antineoplastic Agents; D059005:Topoisomerase II Inhibitors; D000677:Amsacrine