Methodology
The use of caspase inhibitors in pulsed-field gel electrophoresis may improve the estimation of radiation-induced DNA repair and apoptosis
1 Translational Research Laboratory - IDIBELL, Institut Català d'Oncologia, L'Hospitalet de Llobregat, Spain
2 Biostatistics & Bioinformatics Unit, Department of Epidemiology and Cancer Registry, Institut Català d'Oncologia, L'Hospitalet de Llobregat, Spain
3 Department of Radiation Oncology, Institut Català d'Oncologia, L'Hospitalet de Llobregat, Spain
4 Department of Medical Oncology, Institut Català d'Oncologia, L'Hospitalet de Llobregat, Spain
5 Department of Radiation Oncology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain
Radiation Oncology 2011, 6:6 doi:10.1186/1748-717X-6-6
Published: 15 January 2011Abstract
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.
