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

2. 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

3. 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

4. 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:

5. 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.

6. 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).

7. 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.

8. AGO1522 - normal human skin fibroblasts
Materials and methods
AGO 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 cells per sample.

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

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

11. 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.
53BP foci/h foci/h foci/h foci/h foci/h
30 min h h h h h
(30.4%) (31.4%) (12.8%) (6.8%) (17.4%)
The speed of DNA repair

12. Results
Irradiation with 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.

13. 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

14. 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

15. 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

16. 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.

17. 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.

18. 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