Bystander responses and low dose exposure: Current evidence and future research requirements

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1 Bystander responses and low dose exposure: Current evidence and future research requirements Kevin M. Prise Centre for Cancer Research & Cell Biology, Queen s University Belfast

2 Outline Definition of bystander responses Mechanisms of bystander responses Recent in vivo data Future requirements

3 Radiation induced bystander response when cells respond to their neighbour(s) being irradiated Bystander Signal? Direct Irradiation (Target cell) Bystander (responding) cell Response which does not follow the standard model of biological effect in direct proportion to energy deposited in nuclear DNA (Non-(DNA)-targeted effect)

4 Non targeted responses and radiation risk Cancer risk from ionising radiation exposure at low dose is calculated by extrapolation from high dose exposure (Atomic Bomb survivors) A linear no threshold model (LNT) is used Evidence for bystander responses being both damaging and protective have been observed in experimental systems Risk LNT Epidemiological risk data Dose Dotted lines show differences from LNT

5 Factors involved in bystander signalling Zhou, Hei et al., 2008, Cancer Res. 68, 2233

6 Direct versus bystander gene expression Gene profile studies show differences between direct and bystander responses Primary lung fibroblasts 4 hours after irradiation (0.5Gy Helium ions) Direct response involves p53 activation as a key response Bystander response involves NFkβ as a key node Ghandi et al., 2009, BMC Medical Genomics 1:63

7 Interactions between transformed and non-transformed cells Transformed cells can be triggered to apoptose by ROS produced from nontransformed cells Activation of NADPH oxidase a key trigger in irradiated cells Effects triggered by low doses and controlled by TGFβ Bauer et al., 2009 IJRB

8 Damage propagation mechanisms in bystander cells What are the key damage and sensing pathways in bystander cells? How does damage propagate in these cells? What are the consequences for survival and proliferation? Does the cellular response predict tissue responses?

9 DNA damage is induced in bystander cells Clustered gamma-h2ax foci in cells targeted with Heions (line, 1 particle / µm) Early gamma- H2AX foci induction in bystander cells 30 min after irradiation Burdak-Rothkamm et al., 2007, Oncogene, 26, 933

10 γh2ax induction in bystander cells induced foci per cell NHA T98G γh2ax foci persist in bystander cells Accumulation of damage in response to persistent ROS time (h) Burdak-Rothkamm et al., 2007, Oncogene, 26, 933

11 Blockage of γh2ax foci induction 30 min after X ray irradiation in NHA by ATM and DNA PK inhibition control 1 Gy 1 Gy + 1 um ATMi/DNA-PKi 1 Gy + 5 um ATMi/DNA-PKi X ray irradiation induces γh2ax foci Combination of the ATM inhibitor CO PY64 and the DNA PK inhibitor NU7026 block direct foci induction Inhibitors courtesy of G Smith, KuDos, Cambridge

12 Induction of γh2ax foci in bystander cells is independent of ATM and DNA PK induced foci per cell Combination of the ATM inhibitor CO PY64 and the DNA PK inhibitor NU7026 do not block foci induction in bystander NHA cells 0 MT MT + ATMi 5uM MT + DNA- Pki 5uM MT + ATMi/DNA- PKi 5uM Inhibitors courtesy of G Smith, KuDos, Cambridge

13 DSB and cell cycle checkpoints From Lobrich and Jeggo, 2007, Nat. Rev Cancer, 7, 861

14 Is there a role for ATR in bystander H2AX phosphorylation? H2AX is phosphorylated by DNA PK and ATM at sites of DNA DSB after ionising radiation ATR recruitment to DNA DSB induced by IR depends on ATM ATR is recruited to chromatin in S phase H2AX phoshorylation by ATR requires replication stress, arrested replication forks Bystander γh2ax foci through ATR at stalled replication forks in S phase? (ROS mediated single strand breaks, base damage?)

15 γh2ax foci in ATR mutated cells after direct irradiation, but not in bystander cells ATR mut, control ATR mut, 1 Gy ATR mut, Medium transfer WT, control WT, 1 Gy ATR Seckel cells courtesy of Penny Jeggo, University of Sussex WT, Medium transfer

16 No bystander γh2ax foci induction in ATR mutated fibroblasts (ATR Seckel cells) induced foci per cell WT ATR mut percentage G1 WT ATR mut S/G2 No bystander foci in ATR mutated fibroblasts Cell cycle distribution similar in ATR mutated and control cells time (h) Burdak-Rothkamm et al., 2007, Oncogene, 26, 933

17 Induction of 53BP1 foci in S phase bystander cells 5 4 T98G cells BP1 foci control BrdU - bystander BrdU - induced foci per cell Fraction of cells control BrdU+ bystander BrdU γh2ax 53BP1 F02-98 htert 48BR htert BP1 foci per cell Both 53BP1 and γh2ax were induced to a similar level in bystander T98G cells. 53BP1 foci in bystander cells depended on functional ATR. 53BP1 bystander foci predominantly occur in BrdU-positive S-phase cells. Burdak-Rothkamm et al., 2008, Cancer Research 68, 7059.

18 ATR dependent induction of ATM S1981P foci 48BR htert F02-98 htert DAPI ATM-S1981P 1 Gy DAPI ATM-S1981P 1 Gy FO2-98 htert 48BR htert bystander bystander induced foci control control 5 0 bystander 1 Gy ATM acts downstream of ATR in bystander cells Burdak-Rothkamm et al., 2008, Cancer Research 68, 7059.

19 Radiosensitivity of ATM / and ATR mutated cells Direct radiation 48BR htert wild type T98G glioma F02-98 ATR mut ATM -/- MO59J DNAPKcs mut Increased radiosensitivity in ATM /, DNA PK mutant and ATR mutant cells Burdak-Rothkamm et al., 2008, Cancer Research 68, 7059.

20 ATM / and ATR mutated cells are rescued from bystander cell killing Survival fraction MO59J DNA-PK mut ATM-/- fibroblasts T98G glioma 48 BR htert ATR wt FO2-98 htert ATR-mut Burdak-Rothkamm et al., 2008, Cancer Research 68, No bystander cell killing in ATM / and ATR mutated cells γh2ax foci induction in ATM / and DNA PK mutated bystander cells Cells can be rescued from cell death if a DNA damage response is not activated

21 DNA damage signalling in bystander cells

22 Bystander responses in tissues Can localised irradiation be performed in tissue models? Does the response of cell culture models predict for tissue response? Microbeam

23 porcine urothelium H&E stained ureter section Lumen 100 μm 100 μm 1 mm Lamina propria Superficial cell layer - differentiated 2-3 intermediate cell layers - semi-differentiated, nondividing Basal cell layer, dividing Belyakov et al., 2004 Cell movement

24 Primary urothelial explants 7 day urothelium outgrowth 10mm Scheme of urothelial explant. Cells in the centre are differentiated (black) and at the periphery are proliferating (grey) Urothelial outgrowth cytokeratin positive Explant growth

25 Bystander effect dose response Mean fraction of damaged cells (0) (3) (10) (20) (44) Number of particles delivered to a single location Belyakov et al., 2004 Tissue models show similar response to cell models

26 Differentiation Markers Section of porcine ureter stained with uroplakin III (Green), a specific marker for terminally differentiated urothelium lumen 10 μm uroplakin III positive cell in the explant outgrowth

27 Bystander-induced differentiation *P< 0.05; ** P< 0.01 Single location in a ureter tissue fragment targeted with 10 helium ions Fraction of Uroplakin III positive cells scored 7 days after irradiation of the tissue fragment Each sample from a different ureter Bars represent multiple explants from the same ureter Belyakov et al., 2004

28 Bystander effect tissue targeting 3-D Human Skin Reconstruct Model Belyakov et al. (2005) Proc. Natl. Acad. Sci. USA 102,

29 Bystander effect range in tissue Apoptotic cells Micronucleated cells Bystander response observed in 3 D tissue Range of up to 1 mm from exposed cells Belyakov et al. (2005) Proc. Natl. Acad. Sci. USA 102,

30 Tissue homeostasis? Stem <40% 60% Transit Differentiated urothelium spontaneous <1% damaged Localised irradiation 80% Stem 20% Transit Differentiated urothelium spontaneous damaged Prise and Belyakov, unpublished + Feedback stimulation of proliferation

31 In vivo studies of bystander responses Partial mouse exposures (head shielded) C57BL/6 BALB/c Measurements of skin and spleen after whole body or shielded exposures (0.01 or 1Gy) DNA damage induction and apoptosis in shielded areas Further work shows increased epigentic responses involving methylation Potential role for mirna species in long range transmission of response Koturbash et al., (2008) IJROBP, 70, 554.

32 Evidence for bystander-induced carcinogenesis in vivo Radiosensitive mouse models PTCH1 mutants (+/ ) predisposed to medulloblastoma and skin tumours Mice irradiated with X ray doses 0.036, 3, 8.3Gy at post natal day 2 Irradiation setup for shielded irradiation Mancuso M. et.al. PNAS 2008;105:

33 Medulloblastoma development in Ptch1+/ mice Significant induction of medulloblastoma in shielded mice (3Gy) 39% relative to whole body (62%) No effect of scattered dose (0.036 Gy) by week 31 Mancuso M. et.al. PNAS 2008;105:

34 Levels and kinetics of apoptosis in exposed and shielded P2 mouse cerebellum Increased levels of apoptosis measured in whole body and shielded areas within 3 hours of irradiation Cell death stimulated a compensatory hyperplasia in the external granule layer Mancuso M. et.al. PNAS 2008;105:

35 Summary Bystander responses are well characterised in a range of cell models Recent data suggest accumulation of damage in bystander cells drive cellular responses Studies with 3 D models show effects on damaging and protective process Recent in vivo data (at high dose) highlights the carcinogenic potential of bystander signalling

36 Future Research Requirements Cell studies need to consider signalling between different cell types Normal pre cancerous tumour cells stem transit differentiated 3 D models provide an opportunity to test interactions or relevance to tissue responses at low dose Further in vivo studies are required in a range of models with emphasis on localised low dose irradiation

37 Acknowledgements Radiation Biology Group, CCRCB Giuseppe Schettino Shahnaz Al-Rashid Susanne Burdak-Rothkamm Karl Butterworth Keeva McClelland Deepu Oommen Rasa Ugenskiene Mihaela Ghita Gaurang Patel Elena Zahrieva Anna Acheva Joy Kavanagh Martin Lawlor NICC/CCRCB Joe O Sullivan Dean Fennell Medical Physics Agency Alan Hounsell Conor McGarry European Commission NIH Collaborations Fiona Lyng, DIT, Dublin Chunlin Shao, Fudan University, China Oleg Belyakov, STUK, Helsinki Kathryn Held, MGH, Boston Laurence Tartier, Institute Curie, Orsay, France. Boris Vojnovic, Gray Cancer Institute, Northwood