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