The use of caspase inhibitors in pulsed-field gel electrophoresis may improve the estimation of radiation-induced DNA repair and apoptosis
Balart, Josep (Hospital Universitari de Bellvitge)
Pueyo, Gemma (Hospital Universitari de Bellvitge)
de Llobet, Lara I. (Hospital Universitari de Bellvitge)
Baro, Marta (Hospital Universitari de Bellvitge)
Sole, Xavi (Hospital Universitari de Bellvitge)
Marin, Susanna (Hospital Universitari de Bellvitge)
Casanovas, Oriol (Hospital Universitari de Bellvitge)
Mesia, Ricard (Hospital Universitari de Bellvitge)
Capella, Gabriel (Hospital Universitari de Bellvitge)
Universitat Autònoma de Barcelona

Data:	2011
Resum:	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. © 2011 Balart et al; licensee BioMed Central Ltd.
Drets:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Llengua:	Anglès
Document:	Article ; recerca ; Versió publicada
Publicat a:	Radiation oncology, Vol. 6 Núm. 1 (15 2011) , p. 6, ISSN 1748-717X

DOI: 10.1186/1748-717X-6-6
PMID: 21235815

8 p, 1.3 MB

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