Mechanisms conferring sensitivity to low dose radiation exposure and consideration of potentially sensitive individuals.
Penny Jeggo
Health Effects of Ionising radiation: Bridging the Experimental and Epidemiological Divide. Georgetown University Conference Centre, May 4 -6 2009 Radiation induces a range of DNA damage of which a DNA double strand break is the most biologically significant. The DSBs may be complex involving additional damages DNA damaggpe response ( DDR) processes 2St2 Strat egi es f or d ef ence: Optimising recovery and triaging DSB
REPAIR SIGNAL TRANSDUCTION NHEJ/HR ATM
cell cycle checkpoint arrest apoptosis Optimises time for repair and Eliminates prevents proliferation of damaged cells damaged cells DSBs are the main lethal lesion induced by ionising radiation- hence cells lackinggpp DSB repair proteins are ver y radiation sensitive Radiosensitivity of cells lacking Artemis or DNA ligase IV The DDR processes aim to achieve: 1) Resistance to the killing effects of radiation damage – enhance survival 2) Limit the genomic instability caused by radiation damage – prevent carcinogenesis. AIMS
•Will overview the damage response processes of relevance to DSB formation.
• Consider the known syndromes where patients have MARKED defects in these processes – RARE and extreme cases but useful to consider.
• Consider whether small or modest changes – which might be more common - might confer sensitivity to low dose/dose rate Damage response to DNA double strand breaks 2 Strategies: DSB
REPAIR SIGNAL TRANSDUCTION NHEJ/HR ATM Non-Homologous End-Joining Xrcc 4 F XL
AMP 1. DSB protection 2. DSB remodelling 3. DSB processing 4. DSB ligation 5. DSB resealed Mutations in DNA ligase IV, XLF, DNA-PKcs and Artemis have been identified in patients – but they are RARE . NHEJ also functions during immune development to rejoin enzyme induced DSBs – all the above patients show immunodeficiency.
NHEJ also function during development to repair endoggyenously induced DSBs – therefore the patients show developmental and growth delay – particularly microcephaly.
One patient was identified from his over response to radioth erapy (clini cal radi osensiti vit y). The ATM signalling mediator hierarchy
ATM 53BP1 ATM
MRN
MDC1 Histone RNF8 Ubiq MRN H2AX phosphorylation RNF168
Patients defective in MRN (Mre11 and Nbs1), ATM and RNF168 have been identified Ataxia telangg(iectasia (A-T) - ATM AT like disorder – Mre11 Nijmegen Breakage syndrome – Nbs1 Riddle Syndrome – RNF168.
All are associated with immunodeficiency Growth and developmental delay Clinical radiation sensitivity (when given radiotherapy) CANCER PREDISPOSITION A-T cells are defective in the repair of a subfraction of DSBs. No IR 0.5 h after 3 Gy IR 24 h after 3 Gy IR γ-H2AX analysis after 2 Gy X-rays
80 25 MRC-5 (WT) HSF2 (WT) 60 20 180BR (LIG4) – essential - leaky AT1BR (AT) 15 CJ179 (Art) - null 40 10 Foci per cell 20 5
0 0 00 4 4 8 8 12 12162024 16 20 24 004812 4 8 12 hours days Artemis and ATM are essential for a subset of DSB repair
•Exposure to low doses does not cause major sensitivity to MOST of the cell lines from these patients although some may accumulate unrepaired DSBs – but these patients are rare. Slow repai r of DSB s may h ave t wo consequences:
1) Some cell types may be activate apoptosis resulting in increased cell turnover – maybe important for certain stem cells.
2) Slow DSB repai r may cause i ncreased mi srepai r – this may be particularly important in increasing cancer pppredisposition. Mouse model for LIG4 syndrome LigIVm/m mice have reduced lymphocytes in peripheral blood consistent with mpaired V(D)J recombination and growth retardation - identical to LIG4 patients
The mutati on resul ts i n parti al emb ryoni c l eth ali ty b ut survi vors grow to ad ul th ood . The heads seem also small (microcephaly seen in patients) THIS IS BECAUSE THE EMBRYONIC NEURONAL STEM CELLS ARE VERY SENSITIVE TO UNREPAIRED DSBS See loss of Haematopoeitic stem cell (HSC) numbers and function with age in LigIVm/m mice ie one sees age dependent stem cell depletion What about Milder mutational changes in DDR proteins.
1) Heterozygotes – can be present at 1 % of populations where there are founder mutations.
2) Common (or less common) polymorphisms may be impacting.
3) Dominant negative mutational changes Heterozygosity in mice, humans and cell lines confers reppppair defects and cancer predisposition • Heterozygosity for DNA ligase IV in a tumour prone mouse strain, ink4a/art-/-, results in an elevated frequency of soft-tissue sarcomas with clonal ampp,lifications, deletions and translocations ( (p,,,Sharpless, Mol Cell, 8, 1187-1196, 2001). • In several studies, heterozygosity for NHEJ proteins confers elevated chromosome breaks or other ppyphenotypes. • NBS and A-T are prevalent in the Slavic and Ashkenazi jewish populations, respectively. Epidemiological studies on these populations has shown increased cancer predisposition Nbs1 and ATM heterozygotes.
M. Swift and J. L. Lukin, Cancer Epidemiol Biomarkers Prev 17, 3188-3192 (2008). D. Thompson et al , J Natl Cancer Inst 97, 813-822 (2005) . J. Nowak et al, Eur J Cancer 44, 627-630 (2008). E. Seemanova, J Natl Cancer Inst 99, 1875-1880 (2007). N. Bogdanova et al Int J Cancer 122, 802-806 (2008).
• One study showed sensitivity to low dose radiation exposure in AT heterozygotes. AT heterozygotes show a DSB repair defect following low dose rate
80 exposure
60 0.5 h after 3 Gy IR AT cells No IR 40
20
0 04812162024
Control fibroblasts AT heterozygotes AT cells
Plateau pppghase Fibroblasts exposed to 100 mg/h for 24 h
Data from Kato TA, Nagasawa H, MW Michael, JB Little and Joel Bedford Radiation Research 166 443-453, 2006 Impact of mild mutational changes.
LigIV Syndrome represents defects in DNA ligase IV.
180BR was a mild patient with no observable clinical features but deve lope dld leu kaem ia an ddidfd died from ra ditidiation mor bidity.
411BR was a more severe patient – who had immunodeficiency, mild microcephaly and developmental delay. They both had the same homozygous mutational change in DNA ligase IV but 411BR had additionally two linked polymorphic changes.
180BR homozygous Brct domains R278H Catalytic site polymorphic thr ile + ala val 411BR homozygous 180BR 411BR developpymentally immunodeficient normal
LX wt N-term R278H N-terminal + R278H
C 250 500 750 1000 250 500 750 1000 250 500 750 1000 250 500 750 1000 Adenylation
Ligation
1) All of the immunodeficient patients had no detectable adenylation activity ie clinical severity correlates with nature of mutational change. 2) N-terminal polymorphisms add to second mutational change Mild polymorphisms or mutations can affect the function of a protein 2-3 fold (ie similar decrease in activity compared to a heterozygote)– this does not have a major impact on protein function – may not give a clear clinical phenotype but aspects of the function mayyp be impaired. Dominant negative mutations – are changes that impair the function of the wild type protein. They may also cause mild loss of protein function. Of relevance: •Mild reduced function may lead to enhanced stem cell tu rnove r du rin g chro nic e xposu r e. •Mild reduced function may enhance the infidelity of repair and enhance carcinogenesis Summary • The DDR (DNA damage response) play a major role in maintaining resistance and genome stability following radiation exposure. • Loss of DDR proteins confers major radiosensitivity and cancer predisposition. • Loss of DDR proteins have been observed in disorders termed DNA damage response disorders. • However, these patients are rare and have defined clinical features. • Heterozygosity for these proteins also confers a detectable phenotype in mouse and cell models including sensitivity to low dose radiation. It also confers cancer predisposition in humans • Mild mutational changes or dominant negative mutational changes in these prot tieins h ave b een d escrib ibdled also – andittid some impact on protein function. • It is likely, but not proven, that patients harbouring such mutational changes may show low dose radiation sensitivity – particularly to cancer induction
The fidelity of DSB repair is VERY important DSB repair is a slow process, which allows for cell cycle progression to occur and also degradation of the DNA ends
Replication in the presence of a DSB is difficult
Mitosis with DSBs will result in loss of genetic material