DNA repair kinetics: the effect of dose and radiation quality R. Ugenskiene Jagiellonian University, Medical College Pathomorphology Department Molecular Pathology Laboratory

The summary of all experimental activities DSBs: Dose response via DSB induction following X-ray, 3He particles and protons irradiation DNA repair kinetics: the effect of dose and radiation quality Non-radiation relevant factors, which influence the number of DSBs: The effect of different DSB markers The impact of experimental conditions Three- dimensional particle tracks analyzes Micronuclei: Dose response via micronuclei induction in the samples exposed to X-ray, 3He particles and protons Micronuclei formation kinetics following X-ray exposure The effect of dose and radiation quality on micronuclei size

The sequence of the events after the irradiation Introduction The sequence of the events after the irradiation Proliferation, accumulation of damage, additional genetic alterations = Genomic instability Cancer Normal cell division Mutations Micronuclei formation Cell death Mis-repaired DNA Repaired DNA DSB IR DNA repair, Cell cycle delay Un-repaired DNA

Introduction DSB sensing and repairing is achieved by coordinated and well organized work of a big group of proteins. Following the exposure several proteins have been reported to re-localize to nuclear foci. The most well studied are: γ-H2AX, ATM, 53BP1, Rad51, BRCA1 Mre11-Rad50-Nbs1 complex. Al Rashid et al. Cancer Res 2005;65:10810-10821

Introduction Non-homologous end-rejoining is the most important pathway for radiation-induced DSB repair It is supposed that DSB repair kinetics depends on: radiation quality dose of ionizing radiation efficiency of cell’s DNA repair system.

The aim of work In more details, we aimed to: To analyze the effect of dose and different radiation qualities on double strand break repair kinetics. In more details, we aimed to: To analyze DSB repair kinetics in the samples exposed to 0.1 Gy and 0.25Gy of X-ray (the effect of dose) To follow the DNA repair in the samples exposed to X-ray, 3He particles or protons (the effect of radiation quality).

Technical parameters of irradiation sources Materials and methods Technical parameters of irradiation sources 240 kV X–ray machine (Pantak IV) at the Gray Cancer Institute A vertical collimated microbeam at the Gray Cancer Institute, delivering 2 MeV protons and 3.5 MeV 3He, with targeting accuracy of ±2 μm in 95% of cell targets. 2 MeV Cracow horizontal, focused proton microprobe from the Van de Graaff accelerator at the Institute of Nuclear Physics, characterized by the beam diameter of about 12 µm and the targeting accuracy of 30 μm in 92% of cell targets.

AGO1522 - normal human skin fibroblasts Materials and methods AGO1522 - normal human skin fibroblasts Cell fixing and staining Following the exposure cells were fixed at different time points to allow the damage repair. Anti-ATM or anti-53BP1 primary antibodies were used in combination with Alexa Flour 568 or Alexa Fluor 488 secondary antibodies. Cells nuclei were counterstained with DAPI. DSBs came to light as red or green foci in the cell nucleus. They were scored manually in 50-100 cells per sample.

Irradiation with 0.25 Gy of X-ray, kinetics of foci disappearance Results Irradiation with 0.25 Gy of X-ray, kinetics of foci disappearance Distribution of foci Results are mean±0.95 confidence intervals of the mean.

Irradiation with 0.25 Gy of X-ray, kinetics of foci disappearance Results Irradiation with 0.25 Gy of X-ray, kinetics of foci disappearance 30 min Control 1 h 2 h 9 h 24 h

Irradiation with 0.1 Gy of X-ray, kinetics of foci disappearance Results Irradiation with 0.1 Gy of X-ray, kinetics of foci disappearance Results are mean±0.95 confidence intervals of the mean. 53BP1 2.3 foci/h 0.6 foci/h 0.2 foci/h 0.1 foci/h 0.04 foci/h 30 min-------------1 h------------3 h------------6 h-------------9 h------------24 h (30.4%) (31.4%) (12.8%) (6.8%) (17.4%) The speed of DNA repair

Results Irradiation with 0.25 Gy or 0.1 Gy of X-ray, kinetics of foci disappearance A B C Fig. 19. X-ray irradiation. DSB repair kinetics after irradiation with the dose of 0.1 and 0.25 Gy. (A) Results are mean±0.95 confidence intervals of the mean. (B) The fraction of residual DSBs. (C) The speed of DNA repair.

Irradiation with 3 3He particles, kinetics of foci disappearance Results Irradiation with 3 3He particles, kinetics of foci disappearance Results are mean±0.95 confidence intervals of the mean. Distribution of foci number

Irradiation with 3 3He particle, kinetics of foci disappearance Results Triangle pattern Irradiation with 3 3He particle, kinetics of foci disappearance Control 30 min 1 h 3 h 9 h 24 h

Irradiation with 9 1H particles, kinetics of foci disappearance Results Irradiation with 9 1H particles, kinetics of foci disappearance Results are mean±0.95 confidence intervals of the mean. Distribution of foci number

Irradiation with 0.1 Gy of X-ray, 9 1H or 3 3H particles, Results Irradiation with 0.1 Gy of X-ray, 9 1H or 3 3H particles, kinetics of foci disappearance A B DSB repair kinetics. (A) Results are mean±0.95 confidence intervals of the mean. (B) The fraction of residual foci/cell.

Conclusions DSB repair was radiation dose-dependent. It was slightly quicker in the samples exposed to 0.25 Gy of X-ray during the entire DNA repair process. The speed of DSB repair partially depended on radiation quality, as DSBs induced by protons were repaired with a similar speed as those following X-ray exposure. Damage repair after targeted 3He irradiation was significantly slower. The most effective DSB repair was following X-ray treatment and the least effective after 3He irradiation, resulting in ≈1.5 % and ≈ 33% of un-repaired DSBs respectively at 24 h time point. 53BP1 and ATM presented with slightly different kinetics of foci disappearance, what suggests slightly different their role in DNA repair process.

Acknowledgments Thank you for your attention! J Stachura J. Lekki, O Veselov, Z. Stachura KM Prise, S. Gilchrist, D. Groombridge, G. Patel, M. Folkard The staffs of molecular pathology and immunocytochemistry laboratories CM UJ, Krakow All colleagues from GCI Thank you for your attention! This study was supported by the 6th Frame Programme of the European Commission Project “Studies on cellular response to targeted single ions using nanotechnology” CELLION, MRTN-CT-2003-503923