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Updated: Jul 29, 2026
Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells
Published on: February 2, 2013
A flow cytometry-based method for a high-throughput analysis of drug-stabilized topoisomerase II cleavage complexes
Marcelo de Campos-Nebel1, Micaela Palmitelli2, Marcela González-Cid2
1Laboratorio de Mutagénesis, Instituto de Medicina Experimental (IMEX), Academia Nacional de Medicina, CONICET, Buenos Aires, Argentina. mnebel@hematologia.anm.edu.ar.
Abstract:
Topoisomerase II (Top2) is an important target for anticancer therapy. A variety of drugs that poison Top2, including several epipodophyllotoxins, anthracyclines, and anthracenediones, are widely used in the clinic for both hematologic and solid tumors. The poisoning of Top2 involves the formation of a reaction intermediate Top2-DNA, termed Top2 cleavage complex (Top2cc), which is persistent in the presence of the drug and involves a 5' end of DNA covalently bound to a tyrosine from the enzyme through a phosphodiester group. Drug-induced Top2cc leads to Top2 linked-DNA breaks which are the major responsible for their cytotoxicity. While biochemical detection is very laborious, quantification of drug-induced Top2cc by immunofluorescence-based microscopy techniques is time consuming and requires extensive image segmentation for the analysis of a small population of cells. Here, we developed a flow cytometry-based method for the analysis of drug-induced Top2cc. This method allows a rapid analysis of a high number of cells in their cell cycle phase context. Moreover, it can be applied to almost any human cell type, including clinical samples. The methodology is useful for a high-throughput analysis of drugs that poison Top2, allowing not just the discrimination of the Top2 isoform that is targeted but also to track its removal. © 2016 International Society for Advancement of Cytometry.
Insights
A new flow cytometry method rapidly quantifies Topoisomerase II (Top2) cleavage complexes (Top2cc) in cancer cells. This technique aids in high-throughput drug screening and analysis of Top2-targeted anticancer therapies.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Topoisomerase II (Top2) is a crucial target in anticancer therapy, with drugs like epipodophyllotoxins, anthracyclines, and anthracenediones widely used.
- Drug-induced Top2 cleavage complexes (Top2cc) are persistent intermediates leading to DNA breaks and cytotoxicity, forming a key mechanism for these therapies.
Purpose of the Study:
- To develop a rapid, high-throughput flow cytometry-based method for analyzing drug-induced Top2cc.
- To enable cell cycle phase-specific analysis and application to diverse human cell types, including clinical samples.
Main Methods:
- Development of a flow cytometry assay to quantify drug-induced Top2cc.
- Utilized immunofluorescence-based detection within a flow cytometry platform for high-cell-number analysis.
Main Results:
- The developed method allows rapid analysis of drug-induced Top2cc in a high number of cells.
- The technique is applicable to various human cell types and can be used with clinical samples.
- The method facilitates high-throughput screening of Top2-poisoning drugs and can discriminate targeted Top2 isoforms.
Conclusions:
- Flow cytometry offers a more efficient alternative to laborious biochemical assays and time-consuming microscopy for Top2cc quantification.
- This methodology supports high-throughput drug discovery and characterization of Top2-targeted anticancer agents.
- The assay can track Top2 isoform targeting and drug removal, advancing cancer therapy research.
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