Detection and study of the formation and repair of DNA...
Transcript of Detection and study of the formation and repair of DNA...
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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
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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
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Outline
1. Introduction
2. Aims
3. Methodology
4. Results
5. Conclusion
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Introduction
DNA is a critical target of ionizing radiation.
DNA damage
Exogenous Endogenous
Damaging agents
Radiation exposure
Genetic information
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Introduction
Differences between the character of DNA damage caused
by high-LET and low-LET radiation
High-LET radiation Low-LET radiation
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Introduction
Repair
DNA damage
Unrepair
Mutation
Misrepair
Cell survival
Mutated cell
Cell death
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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Thank you for your attention!