Detection and study of the formation and repair of

DNA double-strand breaks after irradiation

Students:

Ioana-Cezara Bucataru

Bianca-Gabriela Zota

Katarína Žirová

Dan Reimitz

Supervisors:

Tatiana Bulanova

Elena Kruglyakova

Laboratory of Radiation Biology (LRB)

JINR Student Practice Dubna, Russia

2016

Joint Institute for Nuclear Research Laboratory of Radiation Biology

• Supervisors: Tatiana Bulanova

Elena Kruglyakova

• Students: Bucataru Ioana Cezara and Zota Bianca

“Al. I. Cuza” University of Iasi

Katarína Žirová

Comenius University in Bratislava

Dan Reimitz

The Czech Technical University in Prague

Outline

1. Introduction

2. Aims

3. Methodology

4. Results

5. Conclusion

Introduction

DNA is a critical target of ionizing radiation.

DNA damage

Exogenous Endogenous

Damaging agents

Radiation exposure

Genetic information

Introduction

Differences between the character of DNA damage caused

by high-LET and low-LET radiation

High-LET radiation Low-LET radiation

Introduction

Repair

DNA damage

Unrepair

Mutation

Misrepair

Cell survival

Mutated cell

Cell death

Aim of this project

Research of the dose-

dependence and kinetic of

formation and elimination of

ionizing radiation-induced foci

on normal human fibroblasts exposed to γ-rays of 60 Co.

Applications of the investigation

of DNA DSBs repair : 1. Diagnostics

2. Nuclear medicine

3. Space radiobiology

Methodology

Preparation

Visualization of induced DSBs co-immunostaining of

γH2AX/53BP1 foci

Fixation of cells at different

times post-irradiation

Acquisition of images fluorescent microscope

Detailed 3D analysis,

quantitative evaluation

γH2AX/53BP1 foci

Deposition of cells from suspension onto glass

slide in Petri dish

Irradiation

Normal human skin fibroblasts

Methodology

Irradiation

Visualization of induced DSBs co-immunostaining of

γH2AX/53BP1 foci

Fixation of cells at different

times post-irradiation

Acquisition of images fluorescent microscope

Detailed 3D analysis,

quantitative evaluation

γH2AX/53BP1 foci

Rocus-M

The laboratory of Nuclear Problems

Preparation Radiation Doses (Gy)

Dose Rate (Gy/min)

LET (keV/μm)

γ-rays of 60Co

1 3 5

0,745 0,3

Methodology

Irradiation

Visualization of induced DSBs co-immunostaining of

γH2AX/53BP1 foci

Fixation of cells at different

times post-irradiation

Acquisition of images fluorescent microscope

Detailed 3D analysis,

quantitative evaluation

γH2AX/53BP1 foci

Time post-irradiation: 1h, 4h, 24h Preparation

Methodology

Irradiation

Visualization of induced DSBs co-immunostaining of

γH2AX/53BP1 foci

Fixation of cells at different

times post-irradiation

Acquisition of images fluorescent microscope

Detailed 3D analysis,

quantitative evaluation

γH2AX/53BP1 foci

Immunostaining

use of an antibody-based method to detect a

specific protein in a sample

gH2AX (FITC - green)

53BP1

(Texas red)

Nuclei

(DAPI - blue) Merged

Preparation

Visualization of induced DSBs co-immunostaining of γH2AX/53BP1 foci

H2AX

DNA DSB

Secondary antibody

conjugated to

fluorescent label

H2AX

Primary antibody

Secondary antibody

53BP1 53BP1

Methodology

Irradiation

Visualization of induced DSBs co-immunostaining of

γH2AX/53BP1 foci

Fixation of cells at different

times post-irradiation

Acquisition of images fluorescent microscope

Detailed 3D analysis,

quantitative evaluation

γH2AX/53BP1 foci

Axio Imager .M2

Methodology

Irradiation

Visualization of induced DSBs co-immunostaining of

γH2AX/53BP1 foci

Fixation of cells at different

times post-irradiation

Acquisition of images fluorescent microscope

Detailed 3D analysis,

quantitative evaluation

γH2AX/53BP1 foci

Merged 3D image of fibroblast nuclei

1h after irradiation with 3 Gy

Preparation

Results

Kinetics of the repair of double-strand breaks

Control

1 4 24

0

5

10

15

20

time post irradiation [h]

№ o

f γH

2A

X/5

3B

P1

fo

ci

pe

r ce

ll a

fte

r 1

Gy

irra

dia

tio

n

Results

Dose response

1Gy 3Gy 5Gy

0

2

4

6

8

10

№ o

f n

uc

lei

No. of γH2AX/53BP1 per cell

Distribution of foci per cell

Conclusion

• The maximum number of γH2AX/53BP1 foci was observed 1 h

after γ-irradiation with 1 Gy.

• The number of DSBs decreased with time and after 24 hours

number of foci reach the control value.

• The obtained curve of dose response has the dependence

close to linear.

• It was observed the efficiently DSBs repair in human fibroblasts

after irradiation with 1 Gy of gamma rays of 60Co.

1h, 1Gy 24h, 1Gy

Thank you for your attention!