High and low LET Biological implications

1
Grundläggande strålningsbiologiDNA-skador och
deras reparation
-DNA-skador, reparation, kromosomaberrationer
Bo Stenerlöw Biomedicinsk strålningsvetenskap Inst
itutionen för onkologi, radiologi och klinisk
immunologi Rudbecklaboratoriet Uppsala universitet
2
Direct and indirect action of ionising radiation
The indirect effect is mediated by radicals in
water close to DNA

• Low LET 30 direct
• High LET 70 direct

(from Hall)
3
JONISERANDE STRÅLNING AVGER INTE ENERGI
KONTINUERLIGT UTAN I DISKRETA ENERGIPAKET SOM KAN
JONISERA MOLEKYLER
1 Gy
0.00024 C
?-photons
?-particles (or accelerated ions)
1000 photons/cell nucleus 1Gy
10 particles/cell nucleus 1Gy
4
Molekylära effekter
DNA är måltavlan
DNA
(SSB)
(DSB)
2 nm
(modified from Steel)
5
Enkelsträngsbrott (SSB) och basskador orsakar
normalt inga problem för cellen. Dessa repareras
snabbt och lätt.
6
Dubbelsträngsbrott (DSB) kan däremot orsaka stora
problem för cellen!
7
The DNA double-strand break (DSB) is the most
critical lesion

• Important processes
• Cell-cycle regulation
• Repair
• Apoptosis

• DSB can also be indued by other agents/processes
• Chemicals
• Enzymes
• Radicals
• SSB during replication -gt
• DSB

(from van Gent et al. 2001)
8
DNA damage detection

• Pulsed-field gel electrophoresis (PFGE)

5.7 Mbp
0 Gy 10 Gy

• Comet assay

(Wojewodzka et al., 2002)
9
DNA damage detection

• Pulsed-field gel electrophoresis (PFGE)
• -the electric field changes direction pulse
  times of 10 min to 3 h

Sensitivity 2-3 Gy
5.7 Mbp


120º angle between fields
10
Pulsed-field gel electrophoresis (PFGE)
Fraction of DNA ltthreshold size is
measured (sometimes called FAR Fraction Activity
Released, based on the 14-C or 3-H labelled DNA)
F 1 - (1 n d k (1 - k/X)) exp(-n d k)
(Blöcher 1990)
-F is the fraction of DNA with sizes less than
the threshold k in bp -n is the number of DSB per
Gy per bp -d is the radiation dose in Gy and X is
the mean size of a chromosome in bp. For a human
genome X is assumed to be 1.3 x 108 bp
11
Two DNA repair pathways in mammalian cells

• Non-homologous end-joining (NHEJ)
• Homologous recombination (HR)

(from van Gent et al., 2001)
12
Normal cells have effective repair of DSB
Repaired by non-homologous end joining (NHEJ)
13
Normal cells have effective repair of DSB
Repaired by non-homologous end joining (NHEJ)

• most important repair process in human cells
• operates through the whole cell-cycle (G1, S, G2)
• fast process with small sequence deletions!

14
Cells lacking DNA-PK cannot rejoin DSB
X
X

• DNA-PK is a key complex in non-homologous
  end-joining

15
Homologous recombination (HR)

• operates in S/G2 of the cell-cycle

16
Mean energy for an ionization 30-35 eV
direct/indirect action
OH
DNA
2 nm
High LET particle
Low LET photon (1 eV/nm)
(100 eV/nm)
sparsely ionising
densely ionising
17
However, high LET radiation induce (i.e. before
repair) only 50 more DSB than photons
?-photons
?-particles (or accelerated ions)
1000 photons/cell nucleus 1Gy 25-30 DSB
10 particles/cell nucleus 1Gy 40 DSB
18
Repair is slower after high LET irradiation
19
Kromosomer kondenserat DNA
Cellkärna
Kromosomerna endast synliga i mikroskop under
mitosfasen i cellcykeln
20
Cellcykeln DNA, kromatin och kromosomer
Mitos (metafasen) DNA kondenserat enskilda
kromosomer kan identifieras
celldelning
30 nm kromatinfiber
vilofas (G1/G0)
30 nm kromatinfiber
Replikation kopiering av DNA
21
Oreparerade eller felreparerade DSB leder till
kromosomaberrationer
Chromosomal change
cell death
Ring chromosome Dicentric chromosome
DNA double-strand break
Cellen klarar inte att dela sig (mitosdöd)
22
Kromosomaberrationer som leder till mitosdöd
(cellen kan inte dela sig)
Dicentrisk kromosom Ringkromosom
(from Hall)
23
Oreparerade eller felreparerade DSB leder till
kromosomaberrationer
Ring chromosome Dicentric chromosome
Translocation Small deletion
Cellen överlever och kan dela sig -carcinogenes
24
D Dicenric chromosome T Translocated
chromosome A Acentric fragment
(from Steel)