AUTHOR=Degenhardt Sarah , Dreffke Kristin , Schötz Urlike , Petersen Cordula , Engenhart-Cabillic Rita , Rothkamm Kai , Dahm-Daphi Jochen , Dikomey Ekkehard , Mansour Wael Yassin 

TITLE=Establishment of a Transformation Coupled in vitro End Joining Assay to Estimate Radiosensitivity in Tumor Cells

JOURNAL=Frontiers in Oncology

VOLUME=10

YEAR=2020

URL=https://www.frontiersin.org/journals/oncology/articles/10.3389/fonc.2020.01480

DOI=10.3389/fonc.2020.01480

ISSN=2234-943X

ABSTRACT=<p>Here, we present a modified <italic>in vitro</italic> end-joining (<italic>EJ</italic>) assay to quantify EJ capacity, accuracy as well as pathway switch to alternative end-joining (Alt-EJ) or single strand annealing (SSA). A novel transformation assay was established to specifically measure circular repair products, which correlate with classical EJ efficiency. The <italic>EJ</italic> assay was validated using EJ-deficient mammalian cell lines (Ku80, DNA-PKcs, LigIV, or XRCC4 mutants). A pathway switch to Alt-EJ and SSA was seen exclusively in Ku-deficient cells. Circular EJ product formation correlated with cell survival and DSB repair capacity after X-irradiation. Investigation of 14 HNSCC cell lines revealed differences in the total EJ capacity but a broader variation in the amount of circular repair products. Sequencing of repair junctions in HNSCC cells demonstrated a predominance of high-fidelity EJ and an avoidance of both Alt-EJ and SSA. A significant correlation was observed between the amount of circular repair products, repair of IR-induced DSB and radiosensitivity. Collectively, these data indicate that the presented <italic>in vitro-EJ</italic>-assay can not only estimate the repair capacity of cancer cells to enable the stratification into radiosensitive or radioresistant, but can also identify repair pathway deregulation such as a switch to Alt-EJ or SSA, which enables tumor targeting.</p>