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Ivashkevich, Alesia (2008): Investigation into the cellular radiosensitivity of the LEC rat and analysis of candidate genes. Dissertation, LMU München: Fakultät für Biologie



Recent work has underlined the importance of animal models in discovery and characterisation of molecular mechanisms determining radiosensitivity and radioresistance. Enhanced sensitivity of LEC rats to ionizing radiation in terms of the acute radiation syndrome was investigated in the present work on the cellular level and compared to that of LE rats. To understand the molecular basis for the increased radiation sensitivity a series of studies were performed, which included the classical clonogenic survival assay, investigation of double strand break repair by means of PFGE and gH2AX evaluation, comet assay for evaluation of repair of single strand breaks and alkaline labile sites, and analysis of cell cycle progression of asynchronous fibroblast population. Survival assay, PFGE, and H2AX analysis were performed in a standardised experimental system - confluent fibroblasts, synchronized in G1 phase of cell cycle, and comet assays were performed in G0 lymphocytes. The data suggests a mild radiosensitization of LEC fibroblasts compared to LE. The results of studies using the selected model did not reflect the degree of animal sensitivity on the molecular level, since values of dose modifying factor (DMF) were much lower in fibroblasts (DMF2 = 1.32) compared to that of animal sensitivity (DMF = 2.36 for bone marrow syndrome (LD50/30) and DMF = 1.95 of intestinal death (LD50/7)). The investigation of DNA repair and cell cycle did not reveal a significant defect in the studied pathways in synchronized fibroblasts and cell cycle progression was not different from wild type cells. The presented data contradict the published LEC cellular phenotype. Of the possible candidate genes, which are located in the radiosensitivity locus, several were further analysed. Among those, Gata-2 appeared to be the most promising of the positional and functional candidates. However, no mutation in the coding sequence could be identified and mRNA expression levels were similar between control and LEC cells. The presented data suggests that radiosensitivity of LEC rats might be attributed to a mechanism specific for certain target tissue, like bone marrow, or enhanced in cell cycle stages other than G0/G1.