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.
Ferguson
L R
LR
Cancer Research Laboratory, University of Auckland Medical School, New Zealand.
Turner
P M
PM
Hart
D W
DW
Tindall
K R
KR
D000677:Amsacrine / Q000633:toxicity*; D000818:Animals; D000970:Antineoplastic Agents / Q000633:toxicity*; D015152:Blotting, Northern; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D009153:Mutagens / Q000633:toxicity*; D009154:Mutation; D016133:Polymerase Chain Reaction; D013866:Thioguanine / Q000378:metabolism
D000970:Antineoplastic Agents; D009153:Mutagens; D000677:Amsacrine; D013866:Thioguanine