Background. The benefits of radiotherapy and cetuximab have encouraged evaluation of cetuximab after radiotherapy. The aims of this study were to preclinically evaluate the efficacy of cetuximab maintenance after radiotherapy and eventually determine its mechanisms of action.Methods. The A431 human carcinoma cell line was treated in culture with fractionated radiotherapy and cetuximab. The surviving cells were injected s.c. into nude mice to mimic microscopic residual disease. The animals were randomized to receive either cetuximab or saline solution. Tumor growth, cell proliferation (Ki-67), microvessel density (MVD), epidermal growth factor receptor (EGFR) and transforming growth factor (TGF-␣) mRNA transcription, and vascular endothelial growth factor (VEGF) secretion were measured.

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BackgroundRadiation-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.MethodsSamples 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.ResultsWe 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.ConclusionsThe 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.