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

• Published in: Frontiers in oncology Vol. 10; p. 1480
• Main Authors: Degenhardt, Sarah, Dreffke, Kristin, Schötz, Urlike, Petersen, Cordula, Engenhart-Cabillic, Rita, Rothkamm, Kai, Dahm-Daphi, Jochen, Dikomey, Ekkehard, Mansour, Wael Yassin
• Format: Journal Article
• Language: English
• Published: Frontiers Media S.A 20.08.2020
• Subjects: classical NHEJ; DSB repair pathway choice; head and neck squamous cell carcinoma; HNSC; in vitro end joining assay; Oncology; radiosensitivity
• ISSN: 2234-943X; 2234-943X
• DOI: 10.3389/fonc.2020.01480

Here, we present a modified in vitro end-joining ( EJ ) 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 EJ 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 in vitro-EJ -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.