Radiation induced bystander effects adaptive responses and low dose risk Carmel Mothersill and Colin Seymour Medical Physics and Applied Radiation Sciences McMaster University, Hamilton, Ontario Canada
Outline Background on the bystander effect Phenomenology Mechanisms Implications for radiation protection Implications for therapy Data gaps and future approaches
Bystander Effect Communicated damage Non-linear dose response History - Clastogenic factors Laboratory methods Targeted irradiation Medium transfer Search for the effector?
Bystander effects - What responses are seen? Apoptosis and other forms of cell death Induction of early response proteins Oxidative stress Proliferation Genomic instability Cytogenetic effects Transformation
Medium transfer bioassay Cell cultures seeded with a large number of cells are exposed to radiation After 1 hr the culture medium is harvested and filtered to remove debris The medium is then transferred to unirradiated reporter cells seeded at cloning density Samples of medium are reserved for calcium and serotonin assay Can be applied to tissues or whole animals using explant technique
Bystander and direct dose survival curves over six orders of magnitude 60 Co 140 120 100 80 60 ICCM Irradiat 40 20 0 0.01 0.1 1 10 100 1000 10000 Dose (m
Direct v bystander effect % clonogenic cell death 100 90 80 70 60 50 40 30 20 10 0 0 0.01 0.03 0.05 0.1 0.3 0.5 2.5 5 initial dir initial By Radiation dose (Gy)
Issues in relation to the central role of DNA damage in radiobiology - possible conflicts? Effects in cells which were never hit but received signals from hit cells No increase in effect with increasing dose, the lowest possible high LET dose to a population- 1 track to one cell or a very low acute low LET dose (3mGy) -turns on the population effect P53 status of the cells not critical therefore the pathway characteristic of DNA damage response may be circumvented in this situation
Factors suggesting a major involvement of DNA direct damage in producing bystander effects Genetic factors are involved in the signaling and response pathways DNA repair deficient cells have very toxic responses to bystander signals Bystander mechanisms seem to drive genomic instability
Different bystander effects depending on repair ability 140 120 100 80 60 0Gy 0.5Gy 5Gy 40 20 0 HPV-G SW48 GS3 1Br3 ATBr1 180Br CHO-K1 XRS 5 XR-1 Burkitts control Cell Line Raji 9 Raji 10
Factors not supporting a direct DNA damage involvement Lack of a classical dose response Induction of large effects in the mgy region Negative effect of dose fractionation No clear effect of neutron irradiation Evidence for effects following EM field exposure
Bystander effects - How are they expressed? Initial mechanism similar to a stress response [ROS elevated] Long-term perpetuation appears to involve genomic instability type mechanisms Final outcome determined mainly by genetic make-up and life-style factors
What is the signal? Nature of the signal is unknown Destroyed by repeated freeze thaw cycles and destroyed by heating, appears to have a very small size (<400 daltons).
Transduction of the response The initial cellular response to the signal in human keratinocytes Induction of 2 min calcium flux within in 10sec of receiving ICCM Longterm (greater than 6hrs) induction of mitochondrial membrane potential collapse and induction of downstream apoptosis steps Longterm induction of oxy-radical production p53 independent
Calcium pulse following addition of 0.5Gy ICCM to cells 1.0 ICM 0 Gy 0.5 Gy 0.8 Fluo 3 / Fura Red 0.6 0.4 0.2 0.0 0.0 0.3 0.5 0.7 0.9 1.1 1.3 1.5 1.7 1.9 2.1 2.3 2.5 2.7 2.9 3.1 3.3 3.5 3.7 3.9 4.1 4.3 4.5 4.7 4.9 5.1 5.3 5.5 5.7 5.9 6.1 6.3 6.5 6.7 6.9 7.1 Time (mins)
Calcium fluorescence following addition of ICCM to cells B D
Mitochondrial membrane depolarisation 0 Gy 0.005Gy
% cells showing increased ROS following ICCM % cells affected 80 70 60 50 40 30 20 10 0 0 Gy 0.5 Gy 5 Gy Dose to progenitors Initial Passage 1 Passage 3 Passage 5 Passage 7
Signal after exposure to ICCM from 0.005Gy irradiated cells 1.2 1 0.8 0.6 0.4 0.2 0 16 24 32 40 48 56 64 72 80 88 96 104 112 120 128 136 144 152 160 168 176 184 192 0 8 Fluo 3 / Fura Red time (s) 1.2 1 0.8 0.6 0.4 0.2 0 14 28 42 56 70 84 98 112 126 140 154 168 182 196 210 224 238 252 266 280 294 308 322 336 350 364 378 392 0 Fluo3 / Fura Red 0.005 Gy medium 0 Gy medium time (s)
Bystander and direct dose survival curves over six orders of magnitude 60 Co with calcium data 140 120 100 80 60 ICCM Irradiat 40 20 No Ca 2+ Ca 2+ 0 0.01 0.1 1 10 100 1000 10000 Dose (m
Calcium homeostasis hypothesis Calcium influx is the first response in the hit cells and in medium recipients Which channels? L channel blockers stop effect Serotonin, l-deprenyl and reserpine all effect calcium homeostasis and all modulate the effect Intracellular calcium homeostasis controlled in mitochondria - role of bcl-2?
Table1 Peak Fluo 3 /Fura Red ratio value in cultures exposed to 0Gy ICCM or 0.5Gy ICCM in the presence of inhibitors of calcium and ROS. An increase in ratio value indicates an increase in calcium. * p<0.01,** p<0.005 0GyICCM 0.5Gy ICCM No inhibitor 0.54 ± 0.01 1.17 ± 0.02 ** EGTA 0.45 ± 0.02 0.46 ± 0.01 Verapamil 0.47 ± 0.02 0.46 ± 0.03 Nifedipine 0.50 ± 0.03 0.49 ± 0.04 Thapsigargin 0.47 ± 0.01 0.72 ± 0.02 * SOD 0.55 ± 0.01 0.51 ± 0.01 Catalase 0.54 ± 0.01 0.55 ± 0.01
Bystander effect of 5HT with 0.5Gy 100 %CE 80 60 40 20 ** 0.5B 5HT 0B 5HT 0 0 2 4 6 8 [drug] micromolar 0.5B 5HT
Bystander effect of reserpine and 0.5Gy 120 100 %CE 80 60 40 20 ** 0.5B res 0B res 0 0 2 4 6 8 0.5B res [drug] micromolar
Cell colonies pretreated with reserpine QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. Control Bystander medium +10nM Reserpine
Serotonin depletion following irradiation, and the bystander effect 140 120 100 80 60 40 20 0 r 2 =0.76 % of control % control 5HT %bystander HPV-G Trout skin C57 mice Reserpine Human kidney T Human Kidney N
Calcium flux induced in HPV-G cells by 8micromolar 5HT
Effect of 5HT 3 receptor inhibitors Zofran and Kitryl, 120 * % of control survival 100 80 60 40 20 * Control 0.5Gy 0 control 100nM Zof 10miM Zof 100nM Kit 10miM Kit drug treatment
The bystander effect Ionizing radiation GJIC connexins Ca 2+ 1 o and 2 o response 5HT bystander factor molecules Receptors? ROS/Nitric oxide/cytokines Biogenic amines???? response Ca 2+ response Ca 2+ Amplification/ Cascade effects? response Ca 2+
Is the effect relevant in vivo?? Evidence from fresh human tissue irradiated ex vivo Evidence from Mice irradiated in vivo to low total body doses Evidence from bloods taken from radiotherapy patients showing variation during therapy Fish model
Methods for detecting signals in tissues Media harvest from exposed explants or whole tissues Detection of signals using reporter cells which are exposed only to media from exposed samples Endpoints include growth,apoptosis, protein expression, calcium fluxes and mitochondrial responses
Measuring bystander response in vitro or in vivo Fresh tissue/organism Explant pieces Culture and irradiation of explants - assay material Harvest culture medium Add to unirradiated clonogenic cell line and determine SF
Human data 300 normal human urothelial samples show wide variation between subjects 50 samples from benign prostate where blood samples from the same patient were available show correlation between response of both tissues Data for radiotherapy patients blood showing changes in bystander effect during therapy New data from nephrectomy patients show normal tissue signals following ex vivo irradiation but none from tumour cells
Explant technique Original tissue explant with cells stained in situ
Mouse data Bladders taken from mice given 0.5 Gy TBI or irradiation to bladder explants ex vivo. CBA/Ca strain is radiation resistant, C57Bl/6 is radiosensitive Apoptotic cascade induced in cells exposed to signals from the sensitive mice only
Calcium ratios in control and 0.5Gy TBI CBA/Ca and C57BL/6 mice 1 0.9 0.8 Medium from unirradiated tissues from both strains 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 CBA/Ca C57BL/6 time (sec) 1 1.6 0.9 0.8 CBA H 0Gy ITCM CBA H 0.5Gy ITCM 1.4 C57Bl 6 0Gy ITCM C57Bl 6 0.5Gy ITCM 1.2 0.7 Fluo 3 / Fura Red 0.6 0.5 0.4 0.3 1 0.8 0.6 0.4 0.2 0.2 0.1 0 0 8 0 16 24 32 40 48 56 64 72 80 0 88 8 96 104 112 120 16 128 24 136 32 144 40 152 48 160 56 168 64 176 72 184 80 192 88 96 104 112 120 128 136 144 152 160 168 176 184 192 time (sec) time (sec)
Real time calcium flux for Control and CBA/Ca mice (A) and C57BL 6 0.5Gy TBI (B) A B
A Mitochondrial membrane potential B decrease in C57BL/6 0.5Gy TBI A B
Fish data Truly truly in vivo!!!!!
What do bystander effects do to radiation protection? Dissociate Dose from effect Effect from harm Harm from risk Enables the concept of a zone of uncertainty where outcome can be assessed relative to the context in which the dose is delivered
So Bystander effects are BAD? Nothing is black and white! Our reporter assay responds by inducing cell death Genetic background predetermines response options Lifestyle factors such as smoking decrease apoptosis following exposure to bystander signals Magnitude of dose is not as important as response to dose Bystander factors following chronic or repeated exposures appear to be very complex Bottom line: effect does not equate with harm
Link to adaptive response Low dose hypersensitivity and bystander effects are mutually exclusive [published data for 13 cell lines) Adaptive response appears to sector with bystander effect -ie get a bystander effect get an adaptive response (4 cell lines tested so far) Pre-treatment of cells with bystander medium induces resistance to an actual dose. Bcl-2 induction appears to be the key.
Therapeutic possibilities Harness the effect to sensitise tumours or to collapse supporting tissue? Use the assay for predictive testing? Integrate the bystander biology into treatment planning? Therapy for non-malignant conditions?
Proposed dose response relationship for radiation-induced effects Yellow arrows indicate mechanistic break points where new, more appropriate, response pathways emerge Effect Zone of linearity New coping mechanism Zone of uncertainty tolerance saturation Dose Natural background
Possible model for expression of bystander effects in biota With intervention points for protective/therapeutic strategies Radiation or other environmental stressor 5HT Ca 2+ 5HT No signal Targeted cell 1 Genotype and environment dependent 1-4 = potential intervention points signal Genomic instability phenotype Anti-apoptotic proteins Recipient cell 3 4 ROS Chance of Life ± mutation Ca 2+ Genotype and environment. dependent 2 Chance of death pro-apoptotic proteins Apoptosis phenotype
Data Gaps Information about the mechanisms involved in SIGNAL GENERATION What determines RESPONSE CHOICE Relevance to low dose RISK MULTIPLE STRESSOR relevance
Acknowledgements In Ireland Dr Fiona Lyng Ms Paula Maguire And in Canada Dr Richard Smith Ms Carol Bucking Dr Michael Kilemade Ms Alicia O Neill Dr Jennifer Lemon Medical collaborators: In Ireland Dr Michael Moriarty Dr Kiaran O Malley Mr John Harney In Canada Mr Anil Kapoor, Dr Aubrey Gilles, Dr Gurmit Singh This work was supported in ~Ireland by the SFI, CEC, contract number FIGH-CT1999-00003 and the Irish CRAB and in Canada by the CRC Chair Programme and the NSERC discovery grant programme