«Radiosensibilité individuelle : une notion ancienne et son avenir» Radiosensibilité: expression génique Christophe Badie, PhD Senior Scientific Group Leader, Cancer Genetics and Cytogenetics Group Biological Effects department Centre for Radiation, Chemical & Environmental Hazards Public Health England United Kingdom
Gene expression and DNA damage Bacteria DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli KENYON CJ and WALKER GC Proc. Natl. Acad. 1980 Yeast Specific Sac. Cerevisiae genes are expressed in response to DNA-damaging agents RUBY SW and SZOSTAK JW Mol Cel biol. 1985 Mammalian cells DNA damage-inducible transcripts in mammalian cells FORNACE AJ et al. Proc. Natl. Acad. Sci. 1988 Modulation of gene expression in Syrian hamster embryo cells following ionizing radiation Woloschak GE et al. Cancer Res. 1990 2
Gene expression and radiation sensitivity Wasted a new mutant of the mouse with abnormalities characteristic of ataxia telangiectasia Shultz LD. et al. Nature 1982 The wasted mutation arose spontaneously on the HRS/J background in 1972 Mutant mice develop progressive paralysis (Neuronal degeneration occurs in the brain and spinal cord) and do not survive beyond 30 days Have immune system abnormalities including a defective response to DNA damage in lymphoid cells Differential effect of ionizing radiation on transcription in repair-deficient and repairproficient mice Munson GP and Woloschak GE. Cancer Res. 1990 wst/wst RNA dot blot examining c-fos gene expression in the gut of B6C3F1, wst/, and wst/wst mice strains in untreated control mice and mice 5 min and 60 min following exposure to 50 cgy (gut dose) of JANUS fission-spectrum neutrons 3
Ionising Radiation Exposure and Gene Expression Exposure of cells to IR activates multiple signal transduction pathways which result in complex alterations in gene expression Out of the ~25,000 human protein-coding genes, a few percent can have there expression changed in response to IR Identification of potential mrna biomarkers in peripheral blood lymphocytes for human exposure to ionizing radiation Amundson et. al International Journal of Radiation Biology, 2000 The response depends on the dose, the dose-rate, the radiation quality, the lapse between stress and analysis, the individual and the tissue analysed, making gene expression analysis complicated but a great source if information Unknown: Dose response, Time course, Low doses, Inter-individual variability 4
Which cells/tissue? In vitro/ex vivo Gene Expression: -Mouse and human blood -Human lymphocytes in culture (extended cultures of T-lymphocytes) Feeders/IL-2/PHA stimulated - Long term cultures - No viral transformation In vivo Gene Expression: -Mouse blood analysis in live animals -Mouse tissues/organs: heart, thymus, spleen, liver, kidney, brain, skin, bone marrow -Human blood samples after external exposure (Radiotherapy) and Internal exposure (Nuclear Medicine) 5
Gene expression following ionising radiation: Identification of biomarkers for dose estimation and prediction of individual response SYLWIA KABACIK, ALAN MACKAY, NARINDER TAMBER, GRAINNE MANNING,PAUL FINNON, FRANCOIS PAILLIER, ALAN ASHWORTH, SIMON BOUFFLER, &CHRISTOPHE BADIE International Journal of Radiation Biology, 2011 Feb;87(2):115-29. 6
fold change in gene expression fold change in gene expression CDKN1A (p21) Role of the dose Transcriptional response: Dose response, 2 hours Role of the time Transcriptional response: Time course, 2 Gy 25 20 15 6 5 4 3 2 1 0 0 30 60 90 120 150 180 210 240 270 300 330 360 390 Time post irradiation, min. 1.4 1.2 1.0 0.8 0.6 0.4 0.2 fold change in gene expression 10 5 0-5 -10 0.1Gy 0.2Gy 0.3Gy 0.4Gy 0.5Gy 1Gy 2Gy 3Gy 4Gy 5Gy Dose Role of the cell-cycle Cultured T-lymphocytes Blood 2 hours 24 hours 2 Gy 4Gy 2 Gy 4 Gy Up-regulated 31 296 100 234 107 13 116 247 Presentation title - edit in Header and Footer Down-regulated 29 0 267 1 113 7 1109 29
Genes of Interest DNA damage DNA Damage Response ATM p53 PCNA DDB2 Transcriptional mechanisms PHPT1 MDM2 SESN1 FAS MYC BBC3 FDXR TIGAR GADD45 CDNK1A CCNG1 CCNB1 Genes of Interest DNA REPAIR RADIATION RESPONSIVE APOPTOSIS CELL CYCLE REGULATION Cellular responses 8
Multiplex quantitative RT-PCR (6 genes) (sensitivity, reproducibility, high-throughput) TaqMan assays Standard curves serial dilutions with PCR products HPRT1 - FAM CCNG1- HEX PUMA Texas Red CDKN1A CY5 PCNA ATTO680 SESN1 ATTO390
Study Objectives Examine ex vivo transcriptional response of DNA damage response genes in human blood after exposure to high and low doses of radiation Explore inter-individual variability 10
Doses responses 2h or 24h post exposure High doses 0.1-4 Gy Low doses 5 100 mgy 11
Biological dosimetry-high doses MAD: Mean Absolute Difference of estimated doses relative to the true doses 12
Fold change relative to control Fold change relative to control Fold change relative to control Low doses - Dose estimation curve (8 donors) Inter-individual variability at low dose validation with 14 donors (100 mgy) CCNG1 Mean Unknown Linear (Mean) DDB2 Mean Unknown Linear (Mean) 5 FDXR Mean Unknown Linear (Mean) 2 1.6 y = 0.0038x + 1 R² = 0.963 2.5 1.5 y = 0.0079x + 1 R² = 0.946 4 3 2 y = 0.0174x + 1 R² = 0.974 1.2 1 0.8 0 25 50 75 100 Dose (mgy) 0.5 0 25 50 75 100 Dose (mgy) 0 0 25 50 75 100 Dose (mgy) Mean 125mGy 95%CL Upper 221mGy St Dev 49 Lower 30mGy Range 50-223mGy Mean 82mGy 95%CL Upper 151mGy St Dev 35 Lower 14mGy Range 40-149mGy Mean 87mGy 95%CL Upper 171mGy St Dev 43 Lower 3mGy Range 2-168mGy
IR-induced DNA Damage Response (DDR) Inter-individual variability-genetic factors DDR activates many pathways - One of them being the ATM/CHK2/p53 pathway The role of the Ataxia telangiectasia gene in the p53, WAF1/CIP1(p21)- and GADD45-mediated response to DNA damage produced by ionising radiation. Artuso M et al. Oncogene 1995 The AT gene product would thus appear to be involved upstream of p53, GADD45 and WAF1/CIP1 (p21) in the signalling of the presence of strand breaks produced by ionising radiation, with this defect in response contributing to the high cancer risk and radiosensitivity observed in this disorder. Nijmegen breakage syndrome cells fail to induce the p53- mediated DNA damage response following exposure to ionizing radiation. Jongmans W et al. Mol Cell Biol. 1997 The transcriptional activation of p21waf1/cip1 mrna was also lower in 12 NBS fibroblast cultures examined 14
Inter-individual variation in response to IR CDKN1A transcriptional response A patient's reaction to radiotherapy is difficult to predict Hypothesis: Underlying genetic factors contribute to the response of irradiated tissues in the body Prospective study of acute skin reactions in breast cancer patients treated by radiotherapy 15
fold change in gene expression Role of inter-individual variability Transcriptional response: 2 Gy-2 hours 10 8 6 CCNB1 CDK1A PUMA 4 2 1-2 -4 PH4b NR11 AT58 healthy breast cancer AT patient 16
Relative gene expression QRT-PCR analysis of T-lymphocytes from breast cancer patients Irradiated T-lymphocytes (2Gy, 2h) CDKN1A as a marker of severe early radiation toxicity 20 15 severe reactor normal reactor 10 5 Prediction of reaction sensitivity>90% 1 1 11 22 Patient number 17
Detection of radiation sensitive individuals Normal range of transcription Transcriptional screen performed on a genetically heterogeneous mouse population: 50% C57BL/6J 50% C3H/HeH A B C Signalling defect Defective DNA damage signalling Failure of p53 activation after IR Reduced/no activation of p53-induced genes Detected in irradiated blood DNA repair defect (mild) Defective DNA repair Prolonged DNA damage signalling Enhanced transcription of p53-induced genes Detected in irradiated blood DNA repair defect (severe) Defective DNA repair Chronic damage to DNA Activation of p53 dependent genes (Abnormal basal level of expression) Detected in non-irradiated blood An instance of clinical radiation morbidity and cellular radiosensitivity, not associated with ataxia-telangiectasia. Plowman PN. et al. Br J Radiol. 1990 Defective repair of DNA double-strand breaks and chromosome damage in fibroblasts from a radiosensitive leukemia patient. Badie C. et al. Cancer Res. 1995 18
Patients with marked (31cases) or mild (28 controls) late adverse reaction to adjuvant breast radiotherapy Variation in lymphocyte radiosensitivity does not necessarily correlate with normal tissue response to radiotherapy. Gene expression analysis can predict of radiation exposure and may help prediction of normal tissue radiosensitivity. 19
Role of genetic factors: Twin studies Human genetic variation contributes significantly to the observed range of radiosensitivity but few quantitative estimates of heritability are available Evidence for significant heritability of apoptotic and cell cycle responses to IR Finnon P et al. Hum Genet 2008 54 pairs, born 1975-1979 (Part of FinnTwin 16 cohort) 38 DZ pairs, 16 MZ pairs MMZ(7), FMZ(9), MDZ(7), FDZ(15), OSDZ(16) Also includes 39 healthy individuals Heritability of cellular responses to IR: 68% cell cycle 59% apoptosis 20
Evidence for high heritability of basal and post IR gene expression PUMA difference X 2.0 PUMA 1.5 1.0 0.5 0.0 0 Gy 2 Gy 0.5 0.4 0.3 0.2 0.1 0.0 MZ DZ NC Estimation of gene expression heritability using bivariate genetic models Results for the gene BBC3 (PUMA ) shows a high heritability at baseline (65% ), which is even higher after radiation treatment (82%) 21
Epigenetic differences arise during the lifetime of monozygotic twins Mapping chromosomal regions with differential DNA methylation in MZ twins Green and red signals indicate hypermethylation and hypomethylation events 3-year-old twins have a very similar distribution of DNA (yellow colour) 50-year-old twin pair shows abundant changes in the pattern of DNA Fraga MF. et al. PNAS, 2005 22
ATM/CHK2/p53 pathway Ionising radiation induces DSB and therefore activates ATM/CHK2/p53 pathway ATM/CHK2/p53 pathway prevents genomic instability and tumorigenesis (Ataxia telangiectasia, Li-Fraumeni syndrome) Epigenetic mechanisms DNA methylation Histone modifications mirna LncRNA Complex, multilayered regulation of eukaryotic gene expression Chek2ing out the p53 pathway: can Puma lead the way? Hollstein M. Cell Cycle, 2011 23
p53 KO (no p53 gene), p53 heterozygotes mice (1 gene copy), wild type (2), p53-tg (3) and p53-tgb (4) mice 24
p53 copy number dependence of cancer incidence Cancer incidence (%) Cancer incidence (%) Cancer incidence 100 Cancer incidence obtained from Garcia-Cao I et al. 2002 80 60 40 y = -20.7x + 99.3 R² = 0.9842 20 0 0 1 2 3 4 p53 gene dose 120 100 80 60 40 20 0 y = -22.407x + 122.75 R² = 0.9754 y = -21.719x + 120.24 R² = 0.9794 0 1 2 3 4 5 Fold of change in gene expression Puma p21 25
Humans Reproducibility of data from blood sampling p21 Donor Exp 1 Exp 2 Exp 3 H2 2.89 4.46 2.36 H7 3.33 2.42 2.77 H8 6.20 3.22 3.85 H9 2.36 1.94 3.42 H12 3.48 2.95 2.58 H14 3.40 3.07 2.31 H17 3.44 2.28 2.46 H19 1.68 2.52 2.19 H20 2.44 2.92 2.33 H29 2.31 2.47 2.88 H30 1.49 2.00 2.00 H31 2.85 3.33 2.88 H33 2.53 3.62 3.14 H34 2.69 3.58 3.19 H43 1.69 2.38 2.52 H48 1.81 2.32 4.18 H50 1.75 2.40 2.17 H53 1.99 3.21 3.34 H54 2.20 3.79 5.00 H56 1.75 3.82 3.01 H57 2.40 3.39 3.40 H58 1.77 3.92 2.86 H59 1.43 3.34 3.04 H60 1.75 2.03 3.48 H61 1.82 2.47 2.66 H62 2.12 1.91 2.12 H63 1.45 1.55 2.89 H64 1.96 5.55 3.83 H65 2.26 1.51 2.93 H66 1.42 2.66 3.35 H67 2.58 2.37 3.23 H68 1.56 2.22 2.99 Mean 2.34 2.86 2.98 SD 0.94 0.88 0.65 26
Fold of change in gene expression Fold of change in gene expression ATM/CHK2/p53 pathway activity PUMA Puma mrna p21 p21 mrna 7 6 PUMA 7 6 p21 5 5 4 4 3 3 2 2 1 1 0 0 1 2 3 4 5 p53 gene dose 0 0 1 2 3 4 5 p53 gene dose Range of ATM/CHK2/p53 pathway activity in humans 27
Future work Study mirna expression Saliva, Urine, Serum (in exosomes) Study LncRNA expression Transcription in individual cells Molecular counting RNA FISH Third generation of PCR (Digital PCR) Deep sequencing 28
Fold change in mirna expression Fold change in mirna expression mirna Class of non-coding RNAs (mall 18-25nt long) Post-transcriptional regulators of gene expression Control expression of around 60% of the human protein-coding genes Involved in almost all physiological and pathological cell functions Homo sapiens 2042 mature sequences, Mus musculus 1281 mirna expression in vivo in the liver (following 2 Gy whole body exposure ) mir-34a: Direct p53 target mir-21: Involved in all steps of tumorigenesis 3.5 3.0 2.5 2.0 1.5 1.0 0.5 mir-34a *** ** C57Bl/6 CBA 2.5 2.0 1.5 1.0 0.5 * mir-21 * C57Bl/6 CBA 0.0 2h 24h 1month 0.0 2h 24h 1month 29
LncRNA expression in response to IR exposure Endogenous level in a control sample Fold change in gene downregulation after 2Gy of X-ray 12 lncrna X 10 8 6 4 2 0 R² = 0.9975 0 1 2 Atm gene copy number Tissue specific expression of lncrna X 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 C57 30
Copy number Copy number Copy number Detecting Fusion transcripts linked to Leukaemia ncounter Leukemia Fusion Gene Expression Assay Chimeric mrna specific recognition 300 200 100 0 y = 265.28x - 3.9775 R² = 0.9973 0 0.2 0.4 0.6 0.8 1 Dilution Kasumi-1 AML-ETO 1000 800 600 400 200 0 200 150 100 50 0 y = 901.01x + 3.9121 R² = 0.9999 0 0.2 0.4 0.6 0.8 1 Dilution y = 168.37x - 2.2394 R² = 0.999 0 0.2 0.4 0.6 0.8 1 Dilution K562 BCR-ABL MV4-11 MLL-AF4 31
RNA FISH Individual cell analysis 0 Gy 2 Gy 4 hours 32
Human Radiosensitivity A report from the Advisory Group on Ionising Radiation (AGIR) March 2013 Established (by NRPB) in 1995 to : review work on the biological and medical effects of ionising radiation relevant to human health in the occupational, public health, medical and environmental fields and advise on research priorities Report available for free download at: http://www.hpa.org.uk/webw/hpaweb&hpawebstandard/hpaweb_c/131713838 1727
Recommendations - I Research is needed to improve understanding of heterogeneity in radiation response and relevance to cancer. Effort is required to identify/validate biomarkers of radiogenic disease. Effort is required to identify genetic risk factors for radiation diseases. Examine epidemiology datasets for interaction of IR and diet, BMI, alcohol, and IR/smoking interaction for cancers other than lung In retrospective risk assessments at doses 100 msv, tobacco smoking history should be taken into account, especially in cases of α-particle exposure of the lung
Recommendations - II Research is needed on interaction of radiation responses and inflammatory responses. Research is required to validate/refute hypothesis on radiation leukaemia risk in translocation cancers. Information on radon/smoking interaction needs to be provided during health/radon remediation campaigns in high radon areas. Information on interaction of smoking with radiation exposure should be made available to radiation workers at recruitment
Implications for Radiation Protection - Ethics The ability to identify sensitive groups or individuals will need careful consideration, especially for occupational exposure. Possible need to consider the justice in protecting small numbers of very high risk individuals In the absence of routine tests, provision of information on risk modifiers and reduction/avoidance should be the main focus.
Acknowledgements PHE colleagues (especially Sylwia Kabacik, Grainne Manning and Paul Finnon) Simon Bouffler (AGIR) Collaborators: Spanish National Cancer Research Centre Ana Ortega-Molina, Alejo Efeyan and Manuel Serrano University of Helsinki Jaakko Kaprio 37