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Using yeast to study resistance to topoisomerase II-targeting drugs
1Developmental Therapeutics Section, Childrens Hospital, Los Angeles, California 90027.
Abstract:
We have developed a system utilizing the yeast Saccharomyces cerevisiae to probe the mechanism of action of anti-topoisomerase II drugs. This system has enabled us to dissect the mechanism of action of these agents. By inducing the overexpression of yeast topoisomerase II or by reducing the level of activity using temperature-sensitive mutations in topoisomerase II, we have demonstrated that conversion of topoisomerase II to a cellular poison plays a critical role in cell killing. We have also constructed other mutations in the yeast TOP2 gene that are resistant to etoposide and amsacrine and determined the DNA sequences for several of the drug-resistant alleles. The mutations that confer drug resistance map to several regions of the TOP2 gene. A mutation of particular interest changes Ser741 to Trp. This mutation results in hypersensitivity to etoposide but does not alter sensitivity to other agents such as mAMSA. We suggest that this mutation defines a site on the TOP2 protein that is involved in drug:protein interactions.
Insights
We developed a yeast system to study anti-topoisomerase II drugs. This research shows that topoisomerase II (TOP2) becomes a cellular poison, crucial for cell death, and identifies specific TOP2 mutations affecting drug resistance.
Area of Science:
- Molecular Biology
- Genetics
- Pharmacology
Background:
- Topoisomerase II (TOP2) is a critical enzyme for DNA replication and transcription.
- Anti-cancer drugs targeting TOP2 are vital chemotherapeutic agents.
- Understanding the precise mechanism of action of these drugs is essential for developing more effective therapies.
Purpose of the Study:
- To investigate the mechanism of action of anti-topoisomerase II drugs using a yeast model.
- To identify specific mutations in yeast TOP2 that confer resistance or hypersensitivity to these drugs.
- To elucidate the role of TOP2 in cellular toxicity induced by these agents.
Main Methods:
- Development of a yeast Saccharomyces cerevisiae system to study TOP2 function.
- Manipulation of yeast TOP2 levels via overexpression and temperature-sensitive mutations.
- Construction and sequencing of drug-resistant TOP2 alleles, including etoposide and amsacrine resistance.
- Analysis of specific mutations, such as Ser741Trp, for altered drug sensitivity.
Main Results:
- Demonstrated that TOP2 conversion to a cellular poison is critical for cell killing by anti-TOP2 drugs.
- Identified and sequenced multiple drug-resistant TOP2 mutations.
- A specific mutation (Ser741Trp) conferred hypersensitivity to etoposide but not mAMSA.
- Drug resistance mutations were mapped to various regions of the TOP2 gene.
Conclusions:
- The yeast system effectively dissects the mechanism of action of anti-topoisomerase II agents.
- TOP2's role as a cellular poison is a key factor in drug-induced cell death.
- Specific mutations in TOP2, like Ser741Trp, can define critical drug-protein interaction sites, aiding in drug design and resistance studies.