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Lookup NU author(s): Dr Elaine Willmore,
Dr Adrian Frank,
Dr Kay Padget,
Dr Michael Tilby,
Professor Caroline Austin
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We have shown that both DNA topoisomerase (topo) 11α and β are in vivo targets for etoposide using a new assay which directly measures topo 11α and β cleavable complexes in individual cells after treatment with topo 11 targeting drugs. CCRF-CEM human leukemic cells were exposed to etoposide for 2 hr, then embedded in agarose on microscope slides before cell lysis. DNA from each cell remained trapped in the agarose and covalently bound topo II molecules from drug-stabilized cleavable complexes remained associated with the DNA. The covalently bound topo II was detected in situ by immunofluorescence. Isoform-specific covalent complexes were detected with antisera specific for either the α or β isoform of topo II followed by a fluorescein isothiocyanate-conjugated second antibody. DNA was detected using the fluorescent stain Hoechst 33258. A cooled slow scan charged coupled device camera was used to capture images. A dose-dependent increase in green immunofluorescence was observed when using antisera to either the α or β isoforms of topo II, indicating that both isoforms are targets for etoposide. We have called this the TARDIS method, for trapped in agarose DNA immunostaining. Two key advantages of the TARDIS method are that it is isoform-specific and that it requires small numbers of cells, making it suitable for analysis of samples from patients being treated with topo II- targeting drugs. The isoform specificity will enable us to extend our understanding of the mechanism of interaction between topo II-targeting agents and their target, the two human isoforms.
Author(s): Willmore, E., Frank, A., Padget, K., Tilby, M.J., Austin, C.A.
Publication type: Article
Publication status: Published
Journal: Molecular Pharmacology
Print publication date: 01/07/1998
ISSN (print): 0026-895X
ISSN (electronic): 1521-0111
PubMed id: 9658192