Topoisomerase II following Exposure to Amsacrine, an Inhibitor of DNA Megabase Pair Deletions in Mutant Mammalian Cells

Amsacrine, [4'-(9-acridinylamino)-methanesulfon-m.anisidide], belongs to the class of cancer chemotherapeutic agents that target DNA topoisomerase II. We show that, over its cytotoxic range, amsacrine is a potent mutagen of the S1 phenotype in the AL (human • hamster) hybrid cell line. By contrast, amsacrine induction of the HPRTphenotype in AL cells is at least two decades less frequent and is not concentration dependent. Such differential mutation frequencies are hypothesized to reflect the concomitant loss of essential genes neighboring the hprt locus. It may be that some amsacrine cytotoxicity is due to the inactivation of essential genes by large deletions. The AL mutation system is well suited for the detection and mapping of mutations which are large deletions because its MIC1 locus, which controls the expression of the selectable cell surface antigen S1, is on a single human chromosome. This human chromosome 11 is in addition to the genome of the Chinese hamster ovary cell and is basically nonessential. Since there are no sister human chromosomes in AL cells, deletions which extend beyond the MIC1 locus may be conveniently and unambiguously mapped. We have detected the presence or absence of 9 different chromosome 11 markers in 48 SImutants cloned from amsacrine-treated cultures. We find that almost all (92%) of the mutants have deletions of at least 1.5--2 megabase pairs in length. The distribution of marker loss frequencies flanking the MIC1 locus does not appear symmetric with respect to distance from that locus. We speculate that amsacrine-induced deletions are mediated by a series of subunit exchanges between overlapping topoisomerase II dimers at the bases of replicons or larger chromosomal structures such as replicon clusters or chromosome minibands.