title: Etoposide metabolites enhance DNA topoisomerase II cleavage near leukemia-associated MLL translocation breakpoints.
abstract: Chromosomal breakage resulting from stabilization of DNA topoisomerase II covalent complexes by epipodophyllotoxins may play a role in the genesis of leukemia-associated MLL gene translocations. We investigated whether etoposide 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 normal homologues of five MLL intron 6 translocation breakpoints was examined in vitro upon incubation with human DNA topoisomerase IIalpha, ATP, and either etoposide, etoposide catechol, or etoposide quinone. Many of the same cleavage sites were induced by etoposide and by its metabolites, but several unique 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 most 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 metabolites induced more cleavage than etoposide at the same sites within a 40 bp double-stranded oligonucleotide containing two of the translocation breakpoints, confirming the results at a subset of the sites. Cleavage assays using the same oligonucleotide substrate in which guanines at several positions were replaced with N7-deaza guanines indicated that the N7 position of guanine is important in metabolite-induced cleavage, possibly suggesting N7-guanine alkylation by etoposide quinone. Not only etoposide, but also its metabolites, enhance DNA topoisomerase II cleavage near MLL translocation breakpoints in in vitro assays. It is possible that etoposide metabolites may be relevant to translocations.
authors.affiliation: Division of Oncology, Department of Pediatrics, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA.
authors.forename: B D
authors.initials: BD
authors.lastname: Lovett
authors.forename: D
authors.initials: D
authors.lastname: Strumberg
authors.forename: I A
authors.initials: IA
authors.lastname: Blair
authors.forename: S
authors.initials: S
authors.lastname: Pang
authors.forename: D A
authors.initials: DA
authors.lastname: Burden
authors.forename: M D
authors.initials: MD
authors.lastname: Megonigal
authors.forename: E F
authors.initials: EF
authors.lastname: Rappaport
authors.forename: T R
authors.initials: TR
authors.lastname: Rebbeck
authors.forename: N
authors.initials: N
authors.lastname: Osheroff
authors.forename: Y G
authors.initials: YG
authors.lastname: Pommier
authors.forename: C A
authors.initials: CA
authors.lastname: Felix
mesh_terms: D002396:Catechols / Q000378:metabolism / Q000494:pharmacology; D019457:Chromosome Breakage; D004249:DNA Damage; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics*; D004795:Enzyme Stability / Q000187:drug effects; D005047:Etoposide / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology*; D011495:Histone-Lysine N-Methyltransferase; D006801:Humans; D007438:Introns / Q000187:drug effects; D007945:Leukemia, Lymphoid / Q000235:genetics*; D007951:Leukemia, Myeloid / Q000235:genetics*; D051788:Myeloid-Lymphoid Leukemia Protein; D009841:Oligonucleotides / Q000378:metabolism; D011519:Proto-Oncogenes; D011809:Quinones / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity / Q000187:drug effects; D014157:Transcription Factors; D014178:Translocation, Genetic / Q000187:drug effects*
chemical_list: D002396:Catechols; D004268:DNA-Binding Proteins; C497422:KMT2A protein, human; D009841:Oligonucleotides; D011809:Quinones; D014157:Transcription Factors; D051788:Myeloid-Lymphoid Leukemia Protein; D005047:Etoposide; D011495:Histone-Lysine N-Methyltransferase; D004250:DNA Topoisomerases, Type II