Amsacrine-induced mutations in AS52 cells.
Amsacrine is an acridine-derived inhibitor of topoisomerase II that intercalates into DNA. We performed a detailed molecular analysis of 6-thioguanine (6-TG)-resistant mutant colonies arising in AS52 cells following Amsacrine treatment. AS52 cells carry a single copy of the bacterial gpt gene, functionally expressed using the SV40 early promoter and stably integrated into the Chinese hamster ovary genome. A 1-hr treatment with 0.1 to 0.5 microM Amsacrine was both cytotoxic and mutagenic, resulting in an average mutant frequency (MF) of 143 x 10(6) at 0.5 microM. Fifty independent 6-TG-resistant colonies were isolated for further study. These clones were initially characterised by PCR to estimate the relative proportion of putative point mutants and deletions or rearrangements; then a subset of mutants was further characterised by Southern blotting, Northern blotting, and DNA sequence analysis. Total deletion of the gpt gene sequences was found in 1 (2%) of the mutants, and 7 (14%) of the mutant clones had altered PCR patterns, suggesting complex deletions or rearrangements. The remaining 42 (84%) mutants had a wild-type PCR profile. Of these, 21 mutants were further analysed by Southern blotting. Interestingly, Southern blotting revealed genomic deletions/rearrangements in 12 of 21 mutants with a wild-type PCR profile. These deletions/rearrangements were further shown to affect gpt gene expression. The remaining nine mutants with a wild-type PCR profile were sequenced. Four of these mutants had mutations in the gpt structural gene. Overall, genomic deletions/rearrangements were observed in 12/21 independent mutants subjected to PCR and Southern blotting. Thus, deletions/rearrangements were the most common mutation observed following Amsacrine treatment of AS52 cells.