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N-terminal and core-domain random mutations in human topoisomerase II alpha conferring bisdioxopiperazine resistance
L H Jensen1, I Wessel, M Møller
1Department of Pathology, Laboratory Center, Righospitalet, Copenhagen, Denmark. lhjensen@rh.dk
Abstract:
Random mutagenesis of human topoisomerase II alpha cDNA followed by functional expression in yeast cells lacking endogenous topoisomerase II activity in the presence of ICRF-187, identified five functional mutations conferring cellular bisdioxopiperazine resistance. The mutations L169F, G551S, P592L, D645N, and T996L confer > 37, 37, 18, 14, and 19 fold resistance towards ICRF-187 in a 24 h clonogenic assay, respectively. Purified recombinant L169F protein is highly resistant towards catalytic inhibition by ICRF-187 in vitro while G551S, D645N, and T996L proteins are not. This demonstrates that cellular bisdioxopiperazine resistance can result from at least two classes of mutations in topoisomerase II; one class renders the protein non-responsive to bisdioxopiperazine compounds, while an other class does not appear to affect the catalytic sensitivity towards these drugs. In addition, our results indicate that different protein domains are involved in mediating the effect of bisdioxopiperazine compounds.
Insights
Mutations in human topoisomerase II alpha can confer resistance to bisdioxopiperazine compounds like ICRF-187. These mutations affect drug response through different mechanisms, highlighting distinct protein domains involved.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Human topoisomerase II alpha is a crucial enzyme for DNA replication and repair.
- Bisdioxopiperazine compounds, such as ICRF-187, are known inhibitors of topoisomerase II.
- Understanding drug resistance mechanisms is vital for developing effective cancer therapies.
Purpose of the Study:
- To identify mutations in human topoisomerase II alpha that confer resistance to ICRF-187.
- To investigate the mechanisms underlying cellular bisdioxopiperazine resistance.
- To explore the role of different protein domains in drug response.
Main Methods:
- Random mutagenesis of human topoisomerase II alpha cDNA.
- Functional expression in yeast cells lacking endogenous topoisomerase II activity.
- Clonogenic assays to measure cellular resistance to ICRF-187.
- In vitro assays with purified recombinant mutant proteins to assess catalytic inhibition.
Main Results:
- Five functional mutations (L169F, G551S, P592L, D645N, T996L) conferring significant ICRF-187 resistance were identified.
- The L169F mutation resulted in a protein highly resistant to ICRF-187 inhibition in vitro.
- Mutations G551S, D645N, and T996L conferred cellular resistance without affecting in vitro catalytic sensitivity.
- Results suggest at least two distinct classes of mutations conferring bisdioxopiperazine resistance.
Conclusions:
- Cellular resistance to bisdioxopiperazines can arise from mutations that either alter drug interaction or affect other cellular processes.
- Different domains within topoisomerase II alpha are involved in mediating the response to bisdioxopiperazine compounds.
- These findings provide insights into drug resistance mechanisms and potential therapeutic strategies targeting topoisomerase II.