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Antimicrobial Agents and Chemotherapy, April 1998, p. 889-894, Vol. 42, No. 4
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Use of a Rapid Throughput In Vivo Screen To Investigate Inhibitors of Eukaryotic Topoisomerase II Enzymes

Timothy R. Hammonds,1 Anthony Maxwell,1 and John R. Jenkins2,*

Department of Biochemistry, University of Leicester, Leicester LE1 7RH,1 and Centre for Mechanisms of Human Toxicity, University of Leicester, Leicester LE1 9HN,2 United Kingdom

Received 29 September 1997/Returned for modification 16 December 1997/Accepted 23 January 1998

Topoisomerase II catalyzes the passage of one DNA helix through another via a transient double-stranded break. The essential nature of this enzyme in cell proliferation and its mechanism of action make it an ideal target for cytotoxic agents. Saccharomyces cerevisiae topoisomerase II has been frequently used as a model for testing potential inhibitors of eukaryotic topoisomerase II as antitumor agents. The standard in vivo method of estimating the sensitivity of S. cerevisiae to the antitopoisomerase drugs is via inhibition or kill curves which rely on viable-cell counts and is labor intensive. We present an alternative to this, a high-throughput in vivo screen. This method makes use of a drug-permeable S. cerevisiae strain lacking endogenous topoisomerase II, which is modified to express either human topoisomerase IIalpha or IIbeta or S. cerevisiae topoisomerase II carried on plasmids. Each modified strain expresses a full-length topoisomerase II enzyme, as opposed to the more commonly used temperature-sensitive S. cerevisiae mutant expressing yeast or yeast/human hybrid enzymes. A comparison of this new method with a plating-and-counting method gave similar drug sensitivity results, with increased accuracy and reduced manual input for the new method. The information generated has highlighted the sensitivities of different topoisomerase II enzymes and isoenzymes to several different classes of topoisomerase II inhibitor.

* Corresponding author. Mailing address: Centre for Mechanisms of Human Toxicity, University of Leicester, Lancaster Rd., Hodgkin Building, Leicester LE1 9HN, United Kingdom. Phone: 116-252-5179. Fax: 116-252-5616. E-mail: jrj1{at}leicester.ac.uk.

Antimicrobial Agents and Chemotherapy, April 1998, p. 889-894, Vol. 42, No. 4
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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