The use of caspase inhibitors in pulsed-field gel electrophoresis may improve the estimation of radiation-induced DNA repair and apoptosis

Abstract

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.

Tumour-infiltrating regulatory T cell density before neoadjuvant chemoradiotherapy for rectal cancer does not predict treatment response

“…We were not able to replicate the finding from previous similar studies that a high density of CD8 + T cells prior to neoadjuvant treatment is associated with good response [ 17 – 19 ]. A key difference between these studies and ours is that we assessed T cell subset densities objectively across entire tissue fragments using image analysis software ( Supplementary Figure 2 ), whereas the studies by Teng et al and Yasuda et al determined T cell subset densities by visual scoring of randomly selected high power fields [ 18 , 19 ]. Anitei et al used image analysis software, but calculated cell density based on the average of the three most infiltrated areas [ 17 ].…”

“…The only clinical or demographic variable significantly associated with T cell subset density was gender, with females having a more pronounced infiltrate of Foxp3 + and CD8 + cells. While an interesting observation, this finding is unlikely to be clinically important since there was no gender-specific difference in CRT response or survival in this, or in previously studied cohorts [ 18 , 19 , 29 , 31 ] (data not shown).…”

“…Infiltration of tumours by T cells is associated with improved prognosis in many cancers [ 12 – 15 ] and may even be more prognostic than histological TNM stage for patients with stage I-III colorectal cancer [ 16 ]. In rectal cancer patients treated with surgery alone, a higher density of total and CD8 + T cells in the central tumour and invasive margin is associated with improved disease-free and overall survival [ 17 ], and in neoadjuvantly-treated rectal cancer, a greater pre-treatment CD8 + T cell infiltrate predicts good response to CRT [ 17 – 19 ]. In contrast, tumour infiltration by CD4 + regulatory T cells (Tregs), which constitutively express the transcription factor forkhead box protein 3 (Foxp3) and negatively regulate immune responses, is often associated with poorer clinical outcome [ 20 – 23 ].…”

The Treatment Outcomes of Olfactory Neuroblastoma Patients With Frontal Lobe Invasion

“…reported that the 5-year OS and disease-free survival rates were 45 and 41% in patients with esthesioneuroblastoma, and surgery combined with radiation therapy achieved the best average survival rate of 65% ( 7 ). Another meta-analysis showed that the 5-year OS stratified by therapy modality was 73% for surgery combined with radiotherapy, 68% for surgery only, 35% for radiotherapy only, and 26% for neither surgery nor radiotherapy, suggesting that the combination of surgery and radiotherapy was the optimal treatment for patients with ONB ( 8 ). However, little is known about the treatment strategies and outcomes for ONB patients with FLI.…”

Adaptive EGF expression sensitizes pancreatic cancer cells to ionizing radiation through activation of the cyclin D1/P53/PARP pathway

“…The IR-induced caspase cascade may also inactivate the poly(ADP-ribose) polymerase (PARP), an enzyme critical for repairing damaged DNA, in order to block DNA repair, thereby promoting IR-induced apoptotic cell death. Indeed, both PARP-1 and PARP-2 knockout mice exhibit severe deficiencies in DNA repair, showing increased sensitivity to alkylating agents or IR (13).…”