Title: A Spatial Study of the Mechanisms of Low Dose Carcinogenesis Using Targeted Irradiation
Funder: Department of Health (England)

Abstract: Background Our current understanding of the effects of radiation is defined by the Linear No Threshold hypothesis (LNT). It has long been considered that biological effects of exposure to ionizing radiation arise as a direct result of damage occurring in directly irradiated cells. It is increasingly recognized, however, that cells that are neighboring directly hit cells, or ‘bystander’ cells, can also express biological responses and genetic damage. Aims The overall aim of this proposal will be to define in-vivo aspects of radiation bystander signaling and its role in carcinogenesis. 1. Determine the extent of low-dose bystander induced radiation carcinogenesis and its spatial dependency in a radiosensitive mouse model using a combination of shielding approaches and advanced image guided radiation exposure. 2. Perform mechanistic studies in 3-D ex-vivo organ explants, using targeted microbeam approaches, to determine key mechanisms of transmission and focusing on the role of DNA repair, connexin expression and the action of purinergic signaling. Research Plan This will use the Patched (Ptch1) mutant mice, characterized by high radiosensitivity and tumor predisposition. 1.1.Dose effect studies. Partial-body irradiation of Ptch1+/- mice with radiation doses down to 10cGy will allow testing RIBE oncogenic effects at doses that are relevant to cancer risks associated with occupational, diagnostic and environmental exposures to radiation. 1.2.Temporal and spatial effects. We will characterize the temporal and spatial effects of in vivo RIBE. Using shielding strategies, we can vary the ratios of unexposed/exposed regions will be analyzed to understand temporal and spatial effects in transmission of bystander signals. Temporal and spatial transmission of damage (DSBs and apoptosis) in the nervous system will be determined in shielded tissues at distance from the irradiated regions. Image-guided radiation approaches will allow specific targeting of distinct regions in vivo under defined conditions. 2.1.Ex-vivo models. We will test whether bystander responses are induced within a primary brain-explant model by targeting individual cells with precision microbeams The microbeam will target specific compartments of actively proliferating and terminally-differentiated cells, allowing assessment of proliferation-dependent effects, DNA damage and apoptosis. Localised dose to these regions will be delivered to cover the range (0.1–2Gy) being used in the in vivo studies. We will then define how these effects are modulated by specific genetic defects in DNA repair, or by cell-to-cell communication by using primary explants from tissues, of: i) wild-type mice; ii) Rad54-/-, DNAPKcs-/- , and PARP-1-/- mice; ATRflox/-Cre-ERT2+ iii) Cx43+/- mice. 2.2.Transmission of purine nucleotides. In vitro studies have shown that nucleoside transport through gap junctions may mediate long-range radiation signals in cultures of human fibroblasts. As purine nucleotides (such as ATP) can act as intercellular signaling molecules and are known to pass across gap junctions and recent work has confirmed a role in vivo . We will test in brain explants the precise spatial dependency that ATP plays as a potential mediator molecule for non-targeted DNA damage. Research team The project will be based at Queen's University Belfast. It will involve a collaboration between; Prof. Kevin Prise (Radiation Biology) with experience the role of low dose bystander signalling in experimental models, Dr Giuseppe Schettino (Radiation Biophysics) with experience in microbeam approaches and Dr Anna Saran ( in vivo radiobiology) who has extensively characterised the ptch mouse model and its response to radiation exposure and will provide access/advice on the animal models. Potential impact This work will provide fundamental information on the shape of the dose-response curve for radiation carcinogenesis in a well-defined in vivo model. It will input into Research Priority A, “Dose-response relationships for cancer induction by ionizing radiation at low doses ( .


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: CANCER AND NEOPLASMS