Investigation into the cellular radiosensitivity of the LEC rat and analysis of candidate genes

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.