The overall objective of this proposal is to investigate the role of poly(ADP-ribose) polymerase (PARP) in the cellular response to ionizing radiation injury. Evidence accumulated from experiments performed by us and others indicates that, although PARP appears to be an element in the G2 checkpoint in mammalian cells, it is not an essential gene for DNA replication or the nucleotide excision pathway for DNA repair. These findings support that the cellular radiation response rely on effective poly(ADP-ribose) metabolism. We hypothesize that the level and mode of action of activated PARP is critical for recovery from ionizing radiation damage to DNA, and that perturbations in the normal regulatory systems for PARP levels and activity by excessive DNA damage result in the potentiation of the apoptosis pathway in the cellular response. Using Ewing's sarcoma (EWS) cells as the primary experimental model system for radiosensitivity and high PARP levels, we propose an experimental approach designed to: 1) study the transcriptional regulation of PARP and determine if it is altered by radiation exposure, 2) identify proteolytic systems involved in the turnover of the PARP gene product, and 3) establish the participation and role of the PARP-turnover proteases in the response to radiation-induced DNA damage and apoptosis. Data from these experiments will identify molecular events associated with the cellular response to ionizing radiation. An important limiting factor in tumor treatment by radiation therapy is the intrinsic radiosensitivity of tumor cells. Understanding the mechanisms of radiation response, particularly those potentiating tumor cell apoptosis, will allow the development of strategies for improving the clinical therapeutic ratio.
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