title: The use of caspase inhibitors in pulsed-field gel electrophoresis may improve the estimation of radiation-induced DNA repair and apoptosis.
abstract: BACKGROUND
  Radiation-induced DNA double-strand break (DSB) repair can be tested by using pulsed-field gel electrophoresis (PFGE) in agarose-encapsulated cells. However, previous studies have reported that this assay is impaired by the spontaneous DNA breakage in this medium. We investigated the mechanisms of this fragmentation with the principal aim of eliminating it in order to improve the estimation of radiation-induced DNA repair.
  
  
  METHODS
  Samples from cancer cell cultures or xenografted tumours were encapsulated in agarose plugs. The cell plugs were then irradiated, incubated to allow them to repair, and evaluated by PFGE, caspase-3, and histone H2AX activation (γH2AX). In addition, apoptosis inhibition was evaluated through chemical caspase inhibitors.
  
  
  RESULTS
  We confirmed that spontaneous DNA fragmentation was associated with the process of encapsulation, regardless of whether cells were irradiated or not. This DNA fragmentation was also correlated to apoptosis activation in a fraction of the cells encapsulated in agarose, while non-apoptotic cell fraction could rejoin DNA fragments as was measured by γH2AX decrease and PFGE data. We were able to eliminate interference of apoptosis by applying specific caspase inhibitors, and improve the estimation of DNA repair, and apoptosis itself.
  
  
  CONCLUSIONS
  The estimation of radiation-induced DNA repair by PFGE may be improved by the use of apoptosis inhibitors. The ability to simultaneously determine DNA repair and apoptosis, which are involved in cell fate, provides new insights for using the PFGE methodology as functional assay.
authors.affiliation: Translational Research Laboratory-IDIBELL, Institut Català d'Oncologia, L'Hospitalet de Llobregat, Spain. jbalart@iconcologia.net
authors.forename: Josep
authors.initials: J
authors.lastname: Balart
authors.forename: Gemma
authors.initials: G
authors.lastname: Pueyo
authors.forename: Lara I
authors.initials: LI
authors.lastname: de Llobet
authors.forename: Marta
authors.initials: M
authors.lastname: Baro
authors.forename: Xavi
authors.initials: X
authors.lastname: Sole
authors.forename: Susanna
authors.initials: S
authors.lastname: Marin
authors.forename: Oriol
authors.initials: O
authors.lastname: Casanovas
authors.forename: Ricard
authors.initials: R
authors.lastname: Mesia
authors.forename: Gabriel
authors.initials: G
authors.lastname: Capella
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology / Q000528:radiation effects*; D061945:Caspase Inhibitors; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects; D004260:DNA Repair / Q000528:radiation effects*; D004307:Dose-Response Relationship, Radiation; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods* / Q000592:standards; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D008297:Male; D051379:Mice; D008819:Mice, Nude; D009369:Neoplasms / Q000235:genetics / Q000473:pathology / Q000532:radiotherapy; D013223:Statistics as Topic; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured
chemical_list: D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors