TNFR2/p75 and TNFR1/p55 interactions in delayed non-targeted effects of radiation in bone marrow derives EPCs

Thesis (M.A.)--Boston UniversityThe central dogma of radiation has been that all biological effects of ionizing radiation occur through direct damage of DNA in the nucleus. There is however evidence dating back to as early as 1950’s which suggests that radiation can cause damage in unirradiated “bystander” cells through mechanisms currently unknown at low dose exposures (Parsons, 1954). This radiobiological bystander effect has since been observed in numerous studies involving microbeam experiments and media transfer experiments, where the observed DNA damage was significantly higher than the amount of cells directly traversed by radiation (Zhou, 2001; Mothersill, 1998). Elevation in the levels of IL-8, TNF-α, FASL, nitric oxide, and reactive oxygen species have been associated with low dose irradiation and are implicated as potential mediators of bystander effect (Narayanan, 1999; Burr, 2010; Rastogi, 2012). TNF-α has two types of receptors p55 and p75 that are part of the mechanism involving NF-κB gene transcription. In our study, we invested the role of TNF-α receptors in the induction of p-H2AX foci (marker of DNA double strand breaks-DBS) in unirradiated mice endothelial progenitor cells (EPCs) post irradiation (IR). One set of EPCs were irradiated with low dose γ-radiation and the media of the cells transferred to corresponding unirradiated EPCs of wild type (WT), p55 knock out (KO), and p75KO mice. Our data showed that at 24 hours, control WT EPCs had the highest p-H2AX foci/cell and gradually decreased 3 and 5 days post media transfer. In contrast, p75KO and p55KO EPCs had lowest count of p-H2AX foci at 24 hours and increased at 3 days. At 5 days post conditioned media (CM) transfer, p55KO continued to have higher p-H2AX foci in an increasing trend whereas p75KO decreased in p-H2AX foci. Our findings showed that in comparison to WT EPCs, the expression of TNFR1/p55 and TNFR2/p75 greatly influenced the pattern of p-H2AX induction over time. ELISA analysis of the γ-irradiated WT and p55KO EPC conditioned media at different time points of the experiment showed that several cytokines and chemokines such as IL-1α, IL-1β, MCP-1, Rantes, and MIP-1α are associated with the increasing trend of p-H2AX foci in p55KO EPCs. Our in vitro mouse recombinant cytokine treatment of p55KO EPCs, using the concentrations of cytokines determined in the ELISA analysis confirmed the ability of TNF-α to induce p-H2AX foci and identified IL-1α as one of the main inducers