Measurement of Drug‐Stabilized Topoisomerase II Cleavage Complexes by Flow Cytometry

Marcelo de Campos Nebel1, Micaela Palmitelli1, Marcela González‐Cid1

1 Laboratorio de Mutagénesis, Instituto de Medicina Experimental (IMEX), Academia Nacional de Medicina
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.48
DOI:  10.1002/cpcy.21
Online Posting Date:  July, 2017
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The poisoning of Topoisomerase II (Top2) has been found to be useful as a therapeutic strategy for the treatment of several tumors. The mechanism of Top2 poisons involves a drug‐mediated stabilization of a Top2‐DNA complex, termed Top2 cleavage complex (Top2cc), which maintains a 5′ end of DNA covalently bound to a tyrosine from Top2 through a phosphodiester group. Drug‐stabilized Top2cc leads to Top2‐linked‐DNA breaks, which are believed to mediate their cytotoxicity. Several time‐consuming or cell type‐limiting assays have been used in the past to study drug‐stabilized Top2cc. Here, we describe a flow cytometry‐based method that allows a rapid assessment of drug‐induced Top2cc, which is suitable for high throughput analysis in almost any kind of human cell. The analyses of the drug‐induced Top2cc in the cell cycle context and the possibility to track its removal are additional benefits from this methodology. © 2017 by John Wiley & Sons, Inc.

Keywords: high‐throughput analysis; human cells; topoisomerase II cleavage complexes

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Table of Contents

  • Introduction
  • Basic Protocol 1: Detection of Stabilized Top2 Cleavage Complexes in Human Cells Following Exposure to Top2 Poisons
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Detection of Stabilized Top2 Cleavage Complexes in Human Cells Following Exposure to Top2 Poisons

  • HL‐60 cell line (ATCC, #CCL‐640)
  • Etoposide (Sigma, cat. no. 33419–42‐0)
  • Ice
  • 1× PHEM buffer (see recipe)
  • Phenylmethanesulfonyl fluoride (PMSF; Sigma)
  • 2× Extraction buffer (see recipe)
  • 4% paraformaldehyde solution (in PBS)
  • 1× Phosphate‐buffered saline (PBS; Gibco, cat. no. 10010031)
  • Blocking buffer (see recipe)
  • Rabbit anti‐Top2α (H‐231, Santa Cruz Biotechnology) or mouse anti‐Top2β (H‐8, Santa Cruz Biotechnology) primary antibodies
  • Alexa Fluor 488‐conjugated goat anti‐rabbit (Life Technologies) or DyLight 488‐conjugated goat anti‐mouse (Thermo Scientific) secondary antibodies
  • RNase A solution
  • Propidium iodide solution
  • RPMI 1640 (with phenol red, sodium bicarbonate, and L‐glutamine)
  • Fetal bovine serum
  • Micropipettes
  • 37°C, 5% CO 2 incubator
  • 1.5‐ml tubes
  • Microcentrifuge
  • Rotating microtube mixer
  • Flow cytometer with at least a blue laser (488 nm)
  • Cell Quest software or any other analysis software
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Literature Cited

Literature Cited
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