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Etoposide targets topoisomerase IIalpha and IIbeta in leukemic cells: isoform-specific cleavable complexes visualized
E Willmore1, A J Frank, K Padget
1Department of Biochemistry and Genetics, The Medical School, The University of Newcastle-upon-Tyne, Newcastle-upon-Tyne NE2 4HH, United Kingdom.
Abstract:
We have shown that both DNA topoisomerase (topo) IIalpha and beta are in vivo targets for etoposide using a new assay which directly measures topo IIalpha and beta cleavable complexes in individual cells after treatment with topo II 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 alpha or beta 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 alpha or beta 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.
Insights
Etoposide targets both DNA topoisomerase (topo) IIalpha and beta in leukemia cells. A new method, TARDIS, allows for isoform-specific detection of these drug-induced DNA topoisomerase complexes in individual cells.
Area of Science:
- Molecular Biology
- Cell Biology
- Pharmacology
Background:
- DNA topoisomerase II (topo II) is a crucial enzyme involved in DNA replication and transcription.
- Etoposide is a widely used chemotherapeutic agent that targets topo II.
- Understanding the specific isoforms targeted by etoposide is essential for optimizing cancer therapy.
Purpose of the Study:
- To investigate whether both DNA topoisomerase IIalpha and IIbeta are in vivo targets of etoposide.
- To introduce and validate a novel method for detecting drug-stabilized topo II cleavable complexes in individual cells.
Main Methods:
- Development of the TARDIS (trapped in agarose DNA immunostaining) method for in situ detection of topo II cleavable complexes.
- Treatment of CCRF-CEM human leukemic cells with etoposide.
- Isoform-specific immunofluorescence detection of DNA topoisomerase IIalpha and IIbeta covalent complexes.
- Quantification of fluorescent signal using a cooled slow scan charged coupled device camera.
Main Results:
- A dose-dependent increase in immunofluorescence was observed for both topo IIalpha and topo IIbeta.
- This indicates that etoposide stabilizes cleavable complexes with both isoforms of DNA topoisomerase II.
- The TARDIS method demonstrated high sensitivity and isoform specificity.
Conclusions:
- Both DNA topoisomerase IIalpha and IIbeta are in vivo targets of etoposide.
- The TARDIS method is a valuable tool for studying the mechanisms of topo II-targeting drugs.
- This technique can be applied to clinical samples for personalized cancer treatment strategies.