Genome Stability Department of Physiology
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1 Cytogenetic biomarkers of ionising radiation exposure: a multiparametric approach M. Prakash HANDE, Ph.D., M.P.H. Genome Stability Laboratory Department of Physiology Yong Loo Lin School of Medicine National University of Singapore Singapore Genome Stability Department of Physiology Telomeres and telomerase DNA damage response and repair Experimental Cancer Therapeutics Radiation Biology/Biodosimetry Toxicogenomics/Environmental Toxicology 1
2 Frequencies of dicentrics and genomic translocations in mouse splenocytes following whole body, 2 Gy X-irradiation d t f Frequency per cell Dicentrics Genomic Translocations D(t)=0.30 e -0.05t T(t)=0.35 e t Days after exposure Hande et al. (1996a) Int. J. Radiat. Biol. 69: Translocations in mouse splenocytes after in vivo X-irradiation analysed by FISH Frequency per 100 cells Reciprocal Non-reciprocal 0.5 Gy 1.0 Gy 2.0 Gy 3.0 Gy Days after exposure Hande and Natarajan (1998) Int. J. Radiat. Biol. 74, Hande and Natarajan (2009) In: Radiation Risk Health Sciences, Springer Japan. Pp
3 Frequencies of micronuclei and fragments in mouse splenocytes following 2 Gy X-irradiation Frequency per 100 cells Days after exposure Hande et al. (1996) Int. J. Radiat.Biol. 70,
4 Detection of intra-chromosomal exchanges in human lymphocytes Boei et al. (1999) Mutat Res 404: MAYAK PRODUCTION ASSOCIATION April 2001 Ozeorsk, Russia 4
5 Past exposure to densely ionising radiation leaves a unique permanent signature in the genome Hande et al (2003) Am J Hum Genet 72: Intrachromosomal aberrations: pericentric, paracentric inversions and intra-arm inversions measured in chromosome 5; Interchromosomal aberrations: Apparently simple translocations between heterologous pair of chromosome Simple versus Complex Chromosomal exchanges in Mayak workers Simple Complex Controls 4/582 0/582 0/582* Reactor Workers Zero Pu, High γ 67/1424 3/1424 7/1424* Moderately exposed Pu workers 10/444 1/444 3/444* Highly exposed Pu workers 82/ / /1414* Aberration Frequency (%) Hande et al Genes Chromosomes Cancer Sep;44(1):1-9 *total aberrations from complex aberrations 5
6 Complex Chromosome Aberrations in Mayak Workers Hande et al Genes Chromosomes Cancer Sep;44(1):1-9 Approach Individual end points were studied independently by different laboratories Few or no attempts were made to study different biomarkers on a single sample Need for a multi-parametric approach on a single sample following irradiation Initiated the work in
7 Ionising Radiation Gamma radiation Heavy Ion Radiation** Whole blood (A) (n=10) γh2ax cronucleus Chromosomal aberration (PNA FISH, mfish) Gene expression Cytokine profile Peripheral blood derived lymphocytes (B) Carbon (whole blood) (C) (n=10) (n=3) γh2ax γh2ax Micronucleus Micronucleus Chromosomal aberration (PNA FISH, mfish) Gene expression mirna expression Proteomic profile Chromosomal aberration (PNA FISH, mfish) Gene expression Iron (whole blood) (D) (n=3) Micronucleus Chromosomal aberration (PNA FISH, mfish) Gene expression Doses : 0.1 Gy, 0.25 Gy, 0.5 Gy, 1 Gy (1.06 Gy/min, 137 Cs source) Time points post irradiation : 2 h and 24 h **National Institute of Radiological Sciences, Chiba, Japan (R Okayasu) Micronuclei detection in mouse/human cells Hande et al. (1996) Int. J. Radiat.Biol. 70, Poonepalli et al. (2005) Cancer Research 65:
8 Single and Two colour FISH Minor satellite Lambda DNA and c-myc gene Copyright 1994 M. Prakash Hande Copyright 1994 M. Prakash Hande Interphase nuclei: Single colour and two colour FISH Frequency of centromere-positive micronucleus in mouse splenocytes Hande et al. (1996) Int. J. Radiat.Biol. 70,
9 PNA FISH in the detection of telomeres Use of PNA FISH with telomere probe in mouse studies RLC translocation Dicentric RLC Translocation Dicentric Blasco, Lee, Hande et al. 1997, Cell 91:25-34 Hande et al J. Cell Biol.144: Di fagagna, Hande et al Nature Genetics Di Fagagna, Hande et al., 2001 Current Biology 9
10 Use of centromere and telomere PNA probes to identify chromosome aberrations in human cells Boei, Vermeulen, Natarajan 2000 Int J Radiat Biol 76: Low et al Int J Biochem Cell Biol 40: Gopalakrishnan et al Genome Integr 1:16 Vinoth et al Stem Cells Dev 17: Banerjee et al 2008 Breast Cancer Res Treat 107: Differential gene expression profile following irradiation: two way hierarchical clustering 0.25 Gy Radiation Dose (Gy) h 24 h 3 ANOVA (p<0.05) ROW : GENE COLUMN : SAMPLE 10
11 Gene expression profile following gamma irradiation PBMCs Whole Blood Principle component analysis REFIRE Knowledgebase Time/dose of known radiation exposure Dose-response following exposure to human peripheral Blood (ex vivo) Biological data from public domains and other literatures Jayapal, Zeegers, Sethu, Banerjee, Hande 2011 Defence Science Journal, Vol. 61, Transcriptomic Knowledgebase Transcriptomics mirna Cytogenetics High-throughput expression profiles of transcriptome, mirna, etc Dose-response clinical endpoints of all parameters Low, medium & highthroughput profiles of radiation exposure Risk estimation, elucidation of underlying mechanism Risk Estimation Following (low-dose) Ionising Radiation Exposure (REFIRE) 11
12 Today REFIRE Knowledgebase - Our Tool Box for Radiation Biology Pilot population Dose, Time MN, CA DNA damage Bioinformatics analysis Networks Biomarkers From a basic toolbox today to Tomorrow Broad population spectrum Optimised Dose, time catalogue Dose,/Time based catalogue of MN and CA Advance complex analysis databases Complex pathways Catalogue of Time / dose specific markers Integrated design, measurements & tools to enable predictable radiation exposure Summary Donor specific effects and individual variation should be taken in to consideration for biomarker identification Gene expression changes may depend on time after exposure than on dose More than one biological response marker may be necessary in biodosimetry Influence of life-style factors Validation of these biomarkers will be done in a larger population and radiotherapy patients 12
13 Study design for CRP To generate calibration curves for gamma radiation in human blood lymphocytes Human PBL 3 apparently healthy donors 0, 0.25, 0.5, 1, 2, 3 and 4 Gy; 137Cs Dicentric and Micronucleus assay Swaminathan Sethu Grace Low Aik Kia Khaw Birendranath Banerjee Dimphy Zeegers Manikandan Jayapal Susan Loong Li Er, National Cancer Centre, SGH, Singapore Rajamanickam Baskar (Radiation Oncology, National Cancer Centre, Singapore) Acknowledgements Defence Innovative Research Programme, Defence Science and Technology Agency, Singapore; IAEA CRP E Ryuichi Okayasu and Takamitsu Kato, NIRS, Chiba 13
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