Establishment of a Transformation Coupled in vitro End Joining Assay to Estimate Radiosensitivity in Tumor Cells
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Establishment of a Transformation Coupled in vitro End Joining Assay to Estimate Radiosensitivity in Tumor Cells. / Degenhardt, Sarah; Dreffke, Kristin; Schötz, Urlike; Petersen, Cordula; Engenhart-Cabillic, Rita; Rothkamm, Kai; Dahm-Daphi, Jochen; Dikomey, Ekkehard; Mansour, Wael Yassin.
In: FRONT ONCOL, Vol. 10, 2020, p. 1480.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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TY - JOUR
T1 - Establishment of a Transformation Coupled in vitro End Joining Assay to Estimate Radiosensitivity in Tumor Cells
AU - Degenhardt, Sarah
AU - Dreffke, Kristin
AU - Schötz, Urlike
AU - Petersen, Cordula
AU - Engenhart-Cabillic, Rita
AU - Rothkamm, Kai
AU - Dahm-Daphi, Jochen
AU - Dikomey, Ekkehard
AU - Mansour, Wael Yassin
N1 - Copyright © 2020 Degenhardt, Dreffke, Schötz, Petersen, Engenhart-Cabillic, Rothkamm, Dahm-Daphi, Dikomey and Mansour.
PY - 2020
Y1 - 2020
N2 - 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.
AB - 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.
U2 - 10.3389/fonc.2020.01480
DO - 10.3389/fonc.2020.01480
M3 - SCORING: Journal article
C2 - 32974177
VL - 10
SP - 1480
JO - FRONT ONCOL
JF - FRONT ONCOL
SN - 2234-943X
ER -
