Analysis of DNA-protein crosslinks induced by high LET radiation

DNA double strand breaks (DSBs) are the major causes of cell death by ionizing radiation. The yield of DSBs decreases with decreasing the oxygen concentration. In contrast, DNA-protein crosslinks (DPCs), another type of lethal lesions, are preferentially formed under hypoxic conditions. Thus, it is likely that both DSBs and DPCs contribute to the lethal events of hypoxic cells upon irradiation. However, it is elusive how much contributions DSBs and DPCs make to the death of irradiated hypoxic cells. This is crucial to understand the radiation response of hypoxic cells in tumors. In this study we will quantify DSBs and DPCs in tumors irradiated with low and high linear energy transfer (LET) radiations and estimate their contributions to the lethal events of hypoxic cells. This year we developed a method to quantify genomic DPCs. The SCCVII tumors in hind legs of C3H/He mice were irradiated with X-rays under normoxic and hypoxic conditions and extracted from mice. DNA was isolated, treated with FITC, and directly analyzed on a fluorometer. Alternatively DNA was analyzed by Western blotting using anti-FITC antibodies. Both fluorescence and Western signals increased with increasing dose. The signals were greater with hypoxic tumors than with normoxic ones, indicating that the preferential formation of DPCs in hypoxic cells. The parallel analysis of DPCs with gel electrophoresis is progress. (author)