Quantification of radiation-induced DNA double strand break repair foci to evaluate and predict biological responses to ionizing radiation

Abstract

Radiation-induced foci (RIF) are nuclear puncta visualized by immunostaining of proteins that regulate DNA double-strand break (DSB) repair after exposure to ionizing radiation. RIF are a standard metric for measuring DSB formation and repair in clinical, environmental and space radiobiology. The time course and dose dependence of their formation has great potential to predict in vivo responses to ionizing radiation, predisposition to cancer and probability of adverse reactions to radiotherapy. However, increasing complexity of experimentally and therapeutically setups (charged particle, FLASH …) is associated with several confounding factors that must be taken into account when interpreting RIF values. In this review, we discuss the spatiotemporal characteristics of RIF development after irradiation, addressing the common confounding factors, including cell proliferation and foci merging. We also describe the relevant endpoints and mathematical models that enable accurate biological interpretation of RIF formation and resolution. Finally, we discuss the use of RIF as a biomarker for quantification and prediction of in vivo radiation responses, including important caveats relating to the choice of the biological endpoint and the detection method. This review intends to help scientific community design radiobiology experiments using RIF as a key metric and to provide suggestions for their biological interpretation.

Understanding the Radiobiological Mechanisms Induced by 177Lu-DOTATATE in Comparison to External Beam Radiation Therapy

“…While the high dose rate used in EBRT intends to overwhelm the DNA repair capacity, the low dose rate applied with 177 Lu-DOTATATE in a protracted exposure gives cells sufficient time to repair a fraction of damage before the creation of subsequent breaks. Consequently, DNA DSB formation is competing with DNA damage repair in this time window [ 23 ], resulting in a challenging distinction from background variability. Additional differences related to distribution pattern as well as size of foci, representing DNA damage complexity, might be expected between 177 Lu-DOTATATE and EBRT and could be addressed by looking at individual cells by radiation-induced foci detection by microscopy.…”

“…Nevertheless, flow cytometry analysis allowed us to semi-quantitatively assess a large number of conditions (cell lines and time points) to have a first general idea on the extent and kinetics differences between 177 Lu-DOTATATE and EBRT in a panel of cancer cell lines. In EBRT-treated cells, elevated γH2AX/pATM levels observed from 72 h post irradiation might represent a combination of residual unrepaired DSBs, but also apoptotic cells which have also been associated with high γH2AX expression [ 23 , 24 ]. Indeed, apoptosis was observed to be already induced at day 3 in our cell lines, namely COLO-677, EJM and MIA-PACA-2 after EBRT.…”

High replication stress and limited Rad51-mediated DNA repair capacity, but not oxidative stress, underlie oligodendrocyte precursor cell radiosensitivity

“…The other important future direction is to address why OPCs are not able to support RAD51 filament formation to the same extent as NSPCs, especially given that RPA filament formation levels are comparable, as are transcription of the RAD51, RAD52, BRCA1 and BRCA2 genes (Figure 6F ) that are RAD51-associated factors important for RAD51 function in DSB repair ( 93 , 94 ). To consolidate this work, it will be important to systematically monitor the mRNA and protein expression of all RAD51-regulatory factors in both the in vitro cell models used here, as well as in vivo mouse brains across early development.…”

Comparison of the Medical Uses and Cellular Effects of High and Low Linear Energy Transfer Radiation

“…DNA damage, especially DSB, rapidly induces the DNA damage response (DDR) pathway [ 133 , 142 ]. The primary pathways of DBS DNA repair are homologous recombination repair (HRR), non-homologous end-joining (NHEJ), and alternative end-joining (alt-EJ) [ 133 , 142 , 143 , 144 ]. These three pathways involve different initiating proteins that recognize DSB and different downstream enzymes ( Table 4 ).…”