detection and study of the formation and repair of dna...
TRANSCRIPT
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!