Enhanced amsacrine-induced mutagenesis in plateau-phase Chinese hamster ovary cells, with targeting of +1 frameshifts to free 3' ends of topoisomerase II cleavable complexes.
Previous work showed that the DNA double-strand cleaving agents bleomycin and neocarzinostatin were more mutagenic in plateau-phase than in log-phase cells. To determine whether topoisomerase II poisons that produce double-strand breaks by trapping of cleavable complexes would, likewise, induce mutations specific to plateau-phase cells, aprt mutations induced by amsacrine in both log-phase and plateau-phase CHO cells were analyzed. The maximum aprt mutant frequencies obtained were 7 x 10(-6) after treatment with 0.02 microM amsacrine in log phase and 27 x 10(-6) after treatment with 1 microM amsacrine in plateau phase, compared with a spontaneous frequency of < 1 x 10(-6). Base substitutions dominated the spectrum of mutations in log-phase cells, but were much less prevalent in plateau-phase cells. Both spectra also included small deletions, insertions and duplications, as well as few large-scale deletions or rearrangements. About 5% of the log-phase mutants and 16% of the plateau-phase mutants were +1 frameshifts, and all but one of these were targeted to potential free 3' termini of cleavable complexes, as determined by mapping of cleavage sites in DNA treated with topoisomerase II plus amsacrine in vitro. Thus, these insertions may arise from templated extension of the exposed 3' terminus by a DNA polymerase, followed by resealing of the strand, as shown previously for acridine-induced frameshifts in T4 phage.
Patteson
K
K
Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298, USA.
Wang
P
P
Povirk
L F
LF
D000228:Adenine Phosphoribosyltransferase / Q000235:genetics; D000677:Amsacrine / Q000494:pharmacology*; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells / Q000187:drug effects*; D002455:Cell Division; D006224:Cricetinae; D004247:DNA / Q000187:drug effects / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005091:Exons / Q000235:genetics; D016368:Frameshift Mutation; D005796:Genes / Q000187:drug effects; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017384:Sequence Deletion; D013379:Substrate Specificity; D059005:Topoisomerase II Inhibitors
D004791:Enzyme Inhibitors; D059005:Topoisomerase II Inhibitors; D000677:Amsacrine; D004247:DNA; D000228:Adenine Phosphoribosyltransferase; D004250:DNA Topoisomerases, Type II