Radiation-induced induced Genomic Instability and Bystander Effects: implications for radiation leukaemogenesis

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1 Radiation-induced induced Genomic Instability and Bystander Effects: implications for radiation leukaemogenesis University of Dundee Medical School Eric G Wright Professor of Experimental Haematology

2 The Haemopoietic System & Target Cells for Leukaemogenesis Most childhood AML Multipotential Haemopoietic Stem Cells Self-maintenance Most adult ALL,AML CML, MDS Most childhood ALL Myeloid commitment Lymphoid commitment Proliferation Differentiation & maturation Platelets monocytes eosinophils Mast cells B-lymphocytes Natural killer cells Erythrocytes neutrophils basophils Dendritic cells T-lymphocytes

3 Biological Effects of Ionizing Radiation

4 Clonal Chromosomal Aberrations in Descendants of Irradiated Haemopoietic Stem Cells

5 Non-Clonal Chromosomal Aberrations in Descendants of Irradiated Haemopoietic Stem Cells Kadhim et.al., 1992, Nature, 355, 738 Kadhim et.al., 1994, Lancet, 344, 987

6 Radiation-induced induced genomic instability in descendants of haemopoietic stem cells cytogenetic aberrations (Non-clonal effects) gene mutations damage/stress responses Cell death and phagocytic clearance

7 Radiation-induced induced Chromosomal Instability Induced and Expressed in vivo Potentially leukaemogenic irradiation time 3 24 months Unstable aberrations 3 4% Stable aberrations 10% 31% (Watson et.al., Int.J.Radiat. Biol., 77, 409, 2001) (Associated with p53 and CDKNA1/p21 expression, nitric oxide generation & apoptosis) (Coates et.al.) N.B. Significant Inter-individual Variation in all endpoints

8 Radiation-induced induced Genomic Instability Effects demonstrated in a wide variety of cell systems Associated with a variety of morphological & functional abnormalities

9 Radiation-induced induced Genomic Instability Effects observed in cells that are not themselves irradiated but are the progeny of irradiated cells A genome-wide process induced at very high (epigenetic) frequencies Response may saturate at low dose Genetic, morphological and functional abnormalities Persists over many cell generations (indefinitely?) Not universally expressed Expression influenced by cell type & genetic factors Expression in vivo but great variability Lesions tend to have characteristics of spontaneous abnormalities Inter-cellular (bystander) mechanisms implicated Mechanisms involving oxidative stress / free radicals implicated

10 Bystander Effects of Ionizing Radiation Effects in more cells than hit by α-particles Media transfer effects Earlier reports of clastogenic factors in blood of exposed individuals Signallingvia medium/plasma (e.g. nitric oxide & cytokines) Signal production Signals Signalling via gap junctions Signal responses

11 Many Radiation-induced induced Bystander Effects Mechanistic link to induced instability Bystander responses may saturate at low dose

12 Bystander-induced Chromosomal Instability Investigate observation that more stem cells than were hit expressed an instability phenotype Instability expressed in descendants of non-irradiated stem cells

13 Chromosomal Instability Induced by Cellular Interactions Instability expressed in descendants of non-irradiated stem cells

14 Genetic Influences on the Expression of Inducible Instability Irradiated Bone marrow % cells exhibiting instability HBM-1 <1 HBM-2 12 HBM-3 <1 HBM-4 15 CBA/H 11 C57BL/6 4 (C57BL/6 x CBA/H) F 1 4 (C57BL/6 x DBA/2) F 1 3 Kadhim et.al.,lancet 344, 987, 1994 Watson et.al., Int.J.Radiat. Biol., 71, 497, 1997 Controls 2%

15 Radiation-induced induced Delayed Apoptosis versus Chromosomal Aberrations Reflect Genetic Influences on cell signalling individual colonies Non-clonal events Genotype-dependent differences in signalling pathways C57BL/6 CBA/Ca & DBA/2 Influence how instability is expressed

16 Damage Signalling and Apoptosis in vivo is Strongly Influenced by Genetic Factors Wallace et.al., Oncogene 20, 3597, 2001 Coates et.al., J.Pathol., 201, 377, 2003

17 Genetics and Radiation-induced induced Signalling Potential for genetic lesion 2 1 ATM p53 p21 Growth arrest Tissue-specific & genetic factors Misrepair Repair 3 DNA-PK Bax Apoptosis Homeostatic responses 1. Directly-induced damage 2. Instability-derived damage 3. Bystander-mediated damage

18 Oxidative Processes & Radiation-induced induced Chromosomal Instabilty in vitro Superoxide production Clutton et.al., Carcinogenesis, 17, 1633, 1996

19 Increased lysosome number and size extensive membrane ruffling Inflammatory-type type Responses in Irradiated Tissues Neutrophil Infiltration lysosomal enzyme activity NO superoxide Superoxide production Myeloperoxidase Responses are genotype-dependent and contribute to whether outcome might be damaging or protective Lorimore et.al.,oncogene 20, 7085, 2001 Lorimore et.al., Oncogene, 22, 7058, 2003

20 Inflammation and Radiation Responses Inflammation implicated in many conditions including malignancy e.g. radiation leukaemogenesis Walburg et al., Int J Cancer, 3, 150, 1968 Yoshida et al., Leuk.Res., , 1993 Gorbunov et al Radiat.Res., 153, 73, 2000 Increases in cardiovascular, gastrointestinal and respiratory system diseases reported in A-bomb survivors Kodama et.al.,j. Epidemiology, 6, S95,1996 Shimizu et.al.,radiat. Res., 152, 374, 1999 Inflammation implicated Neriishi et.al., Int.J.Radiat.Biol., 77, 475, 2001 Hayashi et.al., Int. J. Radiat. Biol., 79, 129, 2003

21 Non-targeted Response Inflammatory-type responses DAMAGE ROS / RNS Signalling Effectors/Cytokines Targeted Response RADIATION EXPOSURE Genetics Cell type Chromosomal abnormalities Gene mutations Excessive cell death Checkpoint & repair responses Signalling Apoptotic responses Adaptation Differentiation Ineffective responses LIFE / DEATH Effective responses Potential pathological effects Tissue homeostasis

22 Radiation and Leukaemia Ionizing radiation unequivocally leukaemogenic Not all equally exposed individuals develop disease Underlying mechanisms not understood Standard risk estimates attribute 34% of childhood / young adult leukaemia to natural radiation 20% β and γ irradiation 14% α irradiation 5% Rn 9% 210 Po

23 Infant /Childhood Leukaemia Initiation Promotion / Progression Frank disease Target cell Potentially leukaemic cell Leukaemic cell Chromosome translocations (majority in utero) Fusion genes Additional genetic lesions Acquired rapidly (probably in utero) in infant ALL Aquired post-natally in most childhood ALL and AML (Latency 1-15 years) Chromosomal translocations and pre-leukemic clones arise at a substantially higher frequency (~100x) before birth than the cumulative incidence or risk of disease.

24 Ionizing Radiation and Leukaemia Initiation Promotion / Progression Frank disease Target cell Potentially leukaemic cell Leukaemic cell New paradigm: Radiation may initiate at exposure by targeted or untargeted effects. Initiation may be post-exposure due to untargeted effects. Radiation may promote pre-existing initiated cells. Conventional paradigm: Targeted DNA damage initiates leukaemogenesis at time of exposure.

25 Acknowledgements Molecular & Cellular Pathology Michael Boylan Changwei Chen Jennifer Chrystal Phil Coates Joanne Fleming Jana Keeler Kirsten Lindsay Sally Lorimore Joanne McIlrath Geri Sandilands Katherine Ball & David Lane Surgery & Molecular Oncology Caroline Evans & Tony Whetton Biomolecular Sciences Carmel Mothersill Medical Physics & Applied Radiation Sciences Unit Former colleagues Radiation & Genome Stability Unit