cytogenetic biomarkers of ionising radiation exposure: a ... · 5 past exposure to densely ionising...
TRANSCRIPT
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 Laboratory @ Department of Physiology
• Telomeres and telomerase • DNA damage response and repair • Experimental Cancer Therapeutics • Radiation Biology/Biodosimetry • Toxicogenomics/Environmental
Toxicology
2
Frequencies of dicentrics and genomic translocations in mouse splenocytes following whole body, 2 Gy X-irradiation
Genomic Translocations Dicentrics
Days after exposure
Freq
uenc
y pe
r cel
l
Hande et al. (1996a) Int. J. Radiat. Biol. 69:437-446
D(t)=0.30 e-0.05t
T(t)=0.35 e-0.006t
0.50
0.00
0.10
0.20
0.30
0.40
-20 0 80 60 20 40 120 100
d t
f
Days after exposure
Freq
uenc
y pe
r 100
cel
ls
Translocations in mouse splenocytes after in vivo X-irradiation analysed by FISH
0 10
20
30
40
50
60
0 50 100 150 200 250
Hande and Natarajan (1998) Int. J. Radiat. Biol. 74, 441-448
Hande and Natarajan (2009) In: Radiation Risk Health Sciences, Springer Japan. Pp 143-149.
0.5 Gy 1.0 Gy 2.0 Gy 3.0 Gy
0
10
20
30
40
50
60
0 50 100 150 200 250
Reciprocal Non-reciprocal
3
Frequencies of micronuclei and fragments in mouse splenocytes following 2 Gy X-irradiation
Freq
uenc
y pe
r 100
cel
ls
Days after exposure
Hande et al. (1996) Int. J. Radiat.Biol. 70, 375-383
4
Detection of intra-chromosomal exchanges in human lymphocytes
Boei et al. (1999) Mutat Res 404: 45-53
April 2001 – Ozeorsk, Russia MAYAK PRODUCTION ASSOCIATION
5
Past exposure to densely ionising radiation leaves a unique permanent signature in the genome
Intrachromosomal aberrations: pericentric, paracentric inversions and intra-arm inversions measured in chromosome 5; Interchromosomal aberrations: Apparently simple translocations between heterologous pair of chromosome
Hande et al (2003) Am J Hum Genet 72:1162-1170
Simple versus Complex Chromosomal exchanges in Mayak workers
0 2 4 6 8 2 4 6 8
Controls
Reactor Workers Zero Pu, High γ
Moderately exposed Pu workers
Highly exposed Pu workers
Aberration Frequency (%)
Simple Complex
82/1414
10/444
67/1424
4/582
41/1414
1/444
3/1424
0/582
88/1414*
3/444*
7/1424*
0/582*
*total aberrations from complex aberrations Hande et al. 2005 Genes Chromosomes Cancer. 2005 Sep;44(1):1-9
6
Complex Chromosome Aberrations in Mayak Workers
Hande et al. 2005 Genes Chromosomes Cancer. 2005 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 2006
7
Ionising Radiation
Gamma radiation
Whole blood (A)
Peripheral blood derived
lymphocytes (B)
Heavy Ion Radiation**
Carbon (whole blood)
(C)
Iron (whole blood)
(D)
(n=10) γH2AX cronucleus Chromosomal aberration (PNA FISH, mFISH) Gene expression Cytokine profile
(n=10) γH2AX Micronucleus Chromosomal aberration (PNA FISH, mFISH) Gene expression miRNA expression Proteomic profile
(n=3) γH2AX Micronucleus Chromosomal aberration (PNA FISH, mFISH) Gene expression
(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, 137Cs 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, 375-383 Poonepalli et al. (2005) Cancer Research 65: 10977-109883
8
Single and Two colour FISH
Interphase nuclei: Single colour and two colour FISH
Minor satellite Lambda DNA and c-myc gene
Cop
yrig
ht ©
199
4 M
. Pra
kash
Han
de
Cop
yrig
ht ©
199
4 M
. Pra
kash
Han
de
Frequency of centromere-positive micronucleus in mouse splenocytes
Hande et al. (1996) Int. J. Radiat.Biol. 70, 375-383
9
PNA FISH in the detection of telomeres
Blasco, Lee, Hande et al. 1997, Cell 91:25-34
Use of PNA FISH with telomere probe in mouse studies
Hande et al. 1999 J. Cell Biol.144: 589-601
Di fagagna, Hande et al. 1999 Nature Genetics
Di Fagagna, Hande et al., 2001 Current Biology
RLC
Dicentric
RLC
translocation
Translocation
Dicentric
10
Use of centromere and telomere PNA probes to identify chromosome aberrations in human cells
Low et al. 2008 Int J Biochem Cell Biol 40:2583 – 2595
Gopalakrishnan et al. 2010 Genome Integr 1:16
Vinoth et al. 20008 Stem Cells Dev 17:599 – 607
Banerjee et al 2008 Breast Cancer Res Treat 107: 25 -31
Boei, Vermeulen, Natarajan 2000 Int J Radiat Biol 76:163-167
Differential gene
expression profile
following irradiation: two
way hierarchical
clustering
0 0.1 0.25 0.5 0 0.1 0.25 0.5
0.25 Gy
24 h
Radiation Dose (Gy)
2 h
3 ANOVA (p<0.05)
ROW : GENE COLUMN : SAMPLE
11
Gene expression profile following gamma irradiation
PBMCs Principle component analysis
Whole Blood
High-throughput expression profiles of
transcriptome, miRNA, etc
Time/dose of known radiation exposure
Dose-response following exposure to
human peripheral Blood (ex vivo)
Dose-response clinical endpoints of all parameters
Biological data from public
domains and other literatures
Low, medium & high-throughput profiles of
radiation exposure
Risk estimation, elucidation of underlying
mechanism
Transcriptomic Knowledgebase
REFIRE Knowledgebase
Transcriptomics miRNA Cytogenetics
Risk Estimation Following (low-dose) Ionising Radiation Exposure (REFIRE)
Jayapal, Zeegers, Sethu, Banerjee, Hande 2011 Defence Science Journal, Vol. 61, 133-137
12
REFIRE Knowledgebase - Our Tool Box for Radiation Biology
Dose, Time
Networks
Bioinformatics analysis
MN, CA DNA damage
Today
Optimised Dose, time catalogue
Complex pathways
Advance complex analysis
databases
Dose,/Time based catalogue
of MN and CA
Tomorrow
Biomarkers
Catalogue of Time / dose
specific markers
From a basic toolbox today to…
Integrated design, measurements & tools to enable predictable radiation exposure
Pilot population
Broad population spectrum
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
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
Acknowledgements
Defence Innovative Research Programme, Defence Science and Technology Agency, Singapore; IAEA CRP E3. 50.08
• 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)
Ryuichi Okayasu and Takamitsu Kato, NIRS, Chiba