investigating the in vitro repair of complex double strand ......dependence of oer on let 1 10 100...
TRANSCRIPT
Molecular basis for the relative biological
effectiveness of densely ionising radiation
Peter O’Neill
Gray Institute for Radiation Oncology & Biology
University of Oxford, UK
●OH e-
H● +
Track structure defines spatial distribution of
energy deposition events
-heterogeneous and homogenous
Chemistry defines types of lesions and yields -oxygen effects
Sub-cellular distribution of damage defined by
ionisation density of the radiation - clusters of lesions (nm scale), clusters of DSB (mm scale), random
distribution
Distribution of DSB and clustered damage
between cells dependent on
- the radiation dose for sparsely ionising radiation
- the fluence for densely ionising radiations
Spatial distribution of events in cells
Damage complexity is largely dependent on
ionisation density of the radiation
30-40% low-LET =
complex
90% high-LET =
complex
Simple DSB
Complex DSB
Clustered damage
1 2 3
0
10
20
30
40
50
60
70
80
90
100
or more
pe
rce
nta
ge
of to
tal
number of lesions in cluster
low LET high LET
Nikjoo, O’Neill, Goodhead, Terrissol,
Int. J. Radiat. Biol., 71, 467-483 (1997);
Friedland et al., Int. J. Radiat. Biol., early on
line (2011)
Maintaining Genomic Stability
DSB
Ionising
Radiation
Free
Radicals
Exogenous
chemical
species
Replication
errors
clustered DNA
damage
base damage
SSB
+
non-homologous
end joining
base
excision
repair
homologous
recombination
Simple
DSB
Complex
DSB
euchromatin
heterochromatin
Lind et al Radiation Research 160, 366-375 (2003)
Surv
ivin
g f
raction
dose/Gy 2 4 6 8
Co MO59K 60
Co MO59J 60
N MO59K 14 7+
N MO59J 14 7+
Co K 60
Co J 60
● N-ions K
● N-ions J
■ □
Co K 60 ■
Deficiency in DNA damage repair: RBE of ~1
Inactivation of the major DNA
DSB repair pathway, leads to
similar radiosensitivity -
independent of LET.
MO59J- inactive
DNA-PKcs
IR-induced DNA damage
IR
SSB DSB
Base
damage
simple 2 or more damages
within 1-2 helical
turns of the DNA
Base excision repair
SSB repair
Non-homologous end joining
Homologous recombination
Single strand annealing
complex
Non-DSB
clustered
damage
IR-induced DSB – different types
double-ended DSB
-prompt DSB
-frank DSB
Non-homologous end joining
one-ended DSB
-replication induced DSB
Homologous recombination
0
0.2
0.4
0.6
0.8
1
1.2
0 4 8 12 16 20 24
Re
lati
ve Y
ield
of
DSB
s
Time (h)
56Fegamma-…
a
g
Double strand breaks
Variation in dynamics of DSB loss following irradiation
56Fe ion
g-radiation
gH2AX PFGE
Do differences in repair kinetics reflect
DSB of different complexity?
Different sub-sets of proteins recruited to some DSB?
Anderson, Harper, Cucinotta, O’Neill.
Radiat. Res. 174, 195-205 (2010)
Jenner, de Lara, O'Neill,Stevens,
Int. J. Radiat. Biol, 64, 265-273 (1993)
6 h
24 h 0.5 h
1Gy 56Fe ions (1 GeV/nu) – DSB detection by gH2AX
Tracks of DSB (gH2AX foci) remain at longer
times - persistence of DSB reflects complexity of DSB
Jakob et al. NAR 39, 6489 (2011); Jeggo et al, EMBO J., 30, 1079 (2011)
Heterochromatin/euchromatin?
Damage complexity- all damage substrates
are not the same
Primer
extension Write off
repair
Cannibalise
from other car
Simple
damage
complex
damage
Very
complex
damage
Dynamics of repair of DSB induced in cells
irradiated at 37 ºC
gH2AX as quantitative marker of DSB?
Role of heterochromatin?
PFGE γ-H2AX
Time (min) 60 120 180
100
% D
SB r
emai
nin
g
50 Complex DSB and/or
heterochromatin DSB
DSB Repair in Absence of DNA-PKcs: 56Fe
0
10
20
30
40
50
60
70
80
90
100
0 4 8 12 16 20 24
Time (h)
% o
f cell
s w
ith
gam
ma
H2
AX
tra
ck
s
MO59J
MO59K
0
5
10
15
20
25
30
35
40
45
50
0 4 8 12 16 20 24
Time (h)
% o
f ce
lls
wit
h R
AD
51
tra
cks
MO59J
MO59K
RAD51 gH2AX (1 Gy) (1 Gy)
0
20
40
60
80
100
0 4 8 12 16 20 24
Time (h)
% o
f ce
lls
wit
h t
rack
s
MO59J
MO59K
HF19
Inactive DNA-PKcs
Active DNA-PKcs
replication
induced DSBs late
S- & G2-phase
Slow
Repair
Some clustered
damage converted to
DSBs at replication
clustered damage complex DSBs
slow
repair
NHEJ
+DNA-PKcs
HR?
Anderson,
Harper,
Cucinotta,
O’Neill.
Radiat. Res.
174, 195-205
(2010)
Hoglund and Stenerlow, Radiation Research
155, 818-825 (2001)
small DNA fragments induced by nitrogen ions
me
an
~2
5 k
bp
Does the attempted repair of
DSB when clustered occur independently?
Does loss of gH2AX foci reflect repair dynamics
of DSB induced by ion-particles?
multiple
DSB
RBE for DSB on LET
Co
-60
gam
ma-
rays
P 2
50
, MeV
LET
0.4
He
25
0 M
eV/u
, LET
1.6
C 2
50
MeV
/u, L
ET 1
3.8
Fe 2
50
MeV
/u, L
ET 2
60
He
1.7
5 M
eV/u
, LET
10
0
C 1
8.3
3 M
eV/u
, LET
10
0
C 8
.33
MeV
/u, L
ET 2
01
Fe 4
14
MeV
/u, L
ET 2
02
C 2
.71
MeV
/u, L
ET 4
42
Fe 1
15
MeV
/u, L
ET 4
42
D. Alloni, A. Campa, M. Belli, G Esposito, L. Mariotti, M. Liotta, W. Friedland, H. Paretzke and A Ottolenghi. Radiation Research 173, 263 (2010)
Calculated number of DSB from DNA fragment distribution
102
101
100
10-1
10-2
10-3
0
20
40
60
80
100L
ET
(ke
V/u
m)
Residual range (mm)Mark A. Hill, Gray Institute
Variation in LET along the path of a charged particle
150 MeV Proton in water
Multiple
DSB
The effect of oxygen
dependence of OER on LET
1 10 100
LET (keV/m)
OE
R
1
2
3
X-rays
OER generally thought
to be slightly lower for
carbon ions than for
proton or photons.
Complexity of DNA damage
decreases under hypoxia
Stewart et al. Radiat. Res. 76, 587 (2011)
2.8
2.0
1.0
OE
R
LET (keV/m) 1 100 10 1000 0.1
RB
E
1
2
3
4
5
6
7
8
Conclusions
Schematic of RBE of cell killing/OER on LET
C H + 6+
Acknowledgements
Jennifer Anderson
Frank Cucinotta
Mark Hill
Luca Mariotti