DNA-dependent protein kinase: effect on DSB repair, G2/M checkpoint and mode of cell death in NSCLC cell lines

Abstract

Purpose: To evaluate the effect of NU7026, a specific inhibitor of DNA-PKcs, on DNA-double strand break (DSB) repair in a cell cycle specific manner, on the G2/M checkpoint, mitotic progression, apoptosis and clonogenic survival in non-small-cell lung carcinoma (NSCLC) cell lines with different p53 status.

Material and methods: Cell cycle progression, and hyperploidy were evaluated using flow cytometry. Polynucleation as a measure for mitotic catastrophe (MC) was evaluated by fluorescence microscopy. DSB induction and repair were measured by constant-gel electrophoresis and γH2AX assay. The efficiency of DSB rejoining during the cell cycle was assessed by distinguishing G1 and G2/M phase cells on the basis of the DNA content in flow cytometry. The overall effect on cell death was determined by apoptosis and the surviving fraction after irradiation with 2 Gy (SF2) assessed by clonogenic survival.

Results: DSB signaling upon treatment with NU7026, as measured by γH2AX signaling, was differently affected in G1 and G2/M cells. The background level of γH2AX was significantly higher in G2/M compared to G1 cells, whereas NU7026 had no effect on the background level. The steepness of the initial dose effect relation at 1 h after irradiation was less pronounced in G2/M compared to G1 cells. NU7026 had no significant effect on the initial dose-effect relation of γH2AX signaling. In comparison, NU7026 significantly slowed down the repair kinetics and increased the residual γH2AX signal at 24 h after irradiation in the G1 phase of all cell lines, but was less effective in G2/M cells. NU7026 significantly increased the fraction of G2/M phase cells upon irradiation. Moreover, NU7026 significantly increased mitotic catastrophe and hyperploidy, as a measure for mitotic failure after low irradiation doses of about 4 Gy, but decreased both at higher doses of 20 Gy. In addition, radiation induced apoptosis increased in A549, H520 and H460 but decreased in H661 upon NU7026 treatment, with a significant reduction of SF2 in all NSCLC cell lines.

Conclusion: Overall, NU7026 significantly influences the cell cycle progression through the G2- and M-phases and thereby determines the fate of cells. The impairment of DNA-PK upon treatment with NU7026 affects the efficiency of the NHEJ system in a cell cycle dependent manner, which may be of relevance for a clinical application of DNA-PK inhibitors in tumor therapy.

Keywords:

• H2AX
• radiation sensitivity
• DNA-PK
• mitotic catastrophe
• cell cycle
• apoptosis

Acknowledgment

The author would like to thank Dr. Sabine Levegrün for proofreading and constructive criticism of the manuscript.

Disclosure statement

The authors report no conflicts of interest.

Additional information

Notes on contributors

Ali Sak

Ali Sak, PhD, is a biologist and head of the working group “molecular radiation oncology”. He works on biological effects of radiation, in vivo and in vitro effects of combined radio-chemotherapy, modulation of epigenetic factors and their effect on the radiation response of tumor cell lines.

Michael Groneberg

Michael Groneberg is a research assistant and an expert in cell culture and microscopic analysis. He is involved in the planning and processing of the experimental settings.

Martin Stuschke

Martin Stuschke is the Chair of the Department of Radiation Therapy at the University Hospital Essen. His research interests are clinical studies with combined therapy schedules involving chemotherapy and radiotherapy and preclinical studies on the role of DNA repair pathways and checkpoint activation in the response of NSCLC to irradiation.