Diminished or inversed dose-rate effect on clonogenic ability in Ku-deficient rodent cells

Abstract

The biological effects of ionizing radiation, especially those of sparsely ionizing radiations like X-ray and γ-ray, are generally reduced as the dose rate is reduced. This phenomenon is known as ‘the dose-rate effect’. The dose-rate effect is considered to be due to the repair of DNA damage during irradiation but the precise mechanisms for the dose-rate effect remain to be clarified. Ku70, Ku86 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are thought to comprise the sensor for DNA double-strand break (DSB) repair through non-homologous end joining (NHEJ). In this study, we measured the clonogenic ability of Ku70-, Ku86- or DNA-PKcs-deficient rodent cells, in parallel with respective control cells, in response to high dose-rate (HDR) and low dose-rate (LDR) γ-ray radiation (~0.9 and ~1 mGy/min, respectively). Control cells and murine embryonic fibroblasts (MEF) from a severe combined immunodeficiency (scid) mouse, which is DNA-PKcs-deficient, showed higher cell survival after LDR irradiation than after HDR irradiation at the same dose. On the other hand, MEF from Ku70−/− mice exhibited lower clonogenic cell survival after LDR irradiation than after HDR irradiation. XR-V15B and xrs-5 cells, which are Ku86-deficient, exhibited mostly identical clonogenic cell survival after LDR and HDR irradiation. Thus, the dose-rate effect in terms of clonogenic cell survival is diminished or even inversed in Ku-deficient rodent cells. These observations indicate the involvement of Ku in the dose-rate effect.

CDKs-mediated phosphorylation of PNKP is required for end-processing of single-strand DNA gaps on Okazaki Fragments and genome stability

“…The procedure for cell cycle distribution analysis was described in our recent publication (Tsuchiya et al, 2021) and appropriately modified for this study. In brief, the nascently synthesized DNA was labeled with 5-ethynyl-2′-deoxyuridine (EdU) and Alexa Fluor 488 azide through Click reaction using a Click-iT EdU Imaging kit (Life Technologies, cat# C10337) and then cells were stained with propidium iodide (PI) using a Cell Cycle Phase Determination Kit (Cayman Chemicals, cat# 10009349) according to the manufacturer’s instruction.…”

The multifaceted functions of DNA‐PKcs: implications for the therapy of human diseases

“… 56 , 65 DNA‐PKcs and Ku70/Ku80 are thought to comprise the dsDNA sensor for DSB repair through NHEJ. 66 In DNA damage response, lysine‐516, lysine‐74, and lysine‐539 of Ku70 can be methylated by the methyltransferase SMYD2, facilitating the recruitment of Ku70/Ku80/DNA‐PKcs complex. The SMYD2 inhibitor treatment results in accumulation of cytosolic DNA, leading to activation of cGAS‐STING pathway.…”

“…DNA‐PKcs can promote an antimicrobial innate immune response to foreign DNA involved in the secretion of IFN, cytokines, and chemokines 56,65 . DNA‐PKcs and Ku70/Ku80 are thought to comprise the dsDNA sensor for DSB repair through NHEJ 66 . In DNA damage response, lysine‐516, lysine‐74, and lysine‐539 of Ku70 can be methylated by the methyltransferase SMYD2, facilitating the recruitment of Ku70/Ku80/DNA‐PKcs complex.…”