Etoposide metabolites enhance DNA topoisomerase II cleavage near leukemia-associated MLL translocation breakpoints

Chromosomal breakage resulting from stabilization of DNA topoisomerase II c ovalent complexes by epipodophyllotoxins may play a role in the genesis of leukemia-associated MLL gene translocations. We investigated whether etopos ide catechol and quinone metabolites can damage the MLL breakpoint cluster region in a DNA topoisomerase II-dependent manner Like the parent drug and the nature of the damage. Cleavage of two DNA substrates containing the nor mal homologues of five MLL intron 6 translocation breakpoints was examined in vitro upon incubation with human DNA topoisomerase Bo, ATP, and either e toposide, etoposide catechol, or etoposide quinone. Many of the same cleava ge sites were induced by etoposide and by its metabolites, but several uniq ue sites were induced by the metabolites. There was a preference for G(-1) among the unique sites, which differs from the parent drug. Cleavage at mos t:sites was greater and more heat-stable in the presence of the metabolites compared to etoposide. The MLL translocation breakpoints contained within the substrates were near strong and/or stable cleavage sites. The metabolit es induced more cleavage than etoposide at the same sites within a 40 bp do uble-stranded oligonucleotide containing two of the translocation breakpoin ts, confirming the results at a subset of the sites. Cleavage assays using the same oligonucleotide substrate in which guanines at several positions w ere replaced with N7-deaza guanines indicated that the N7 position of guani ne is important in metabolite-induced cleavage, possibly suggesting N7-guan ine alkylation by etoposide quinone. Not only etoposide, but also its metab olites, enhance DNA topoisomerase II cleavage near MLL translocation breakp oints in in vitro assays. It is possible that etoposide metabolites may be relevant to translocations.