DNA DAMAGE AND REPAIR IN IRRADIATED BRAIN AND BRAIN TUMO

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The objective of this research is to elucidate the molecular mechanisms responsible for the differences in DNA repair kinetics observed in normal cells (brain, lymphocytes, macrophages) and tumor cells after irradiation or treatment with drugs. Our previous work indicates that (1) normal nondividing neurons repair radiation-induced DNA damage at a much slower rate than dividing brain tumor cells, (2) structural changes in the DNA of neurons can be used to evaluate the long-term effects of brain irradiation, and (3) recovery from potentially lethal damage is a major determinant of radiocurability in 9L brain tumors. We propose to use either the alkaline elution technique of Kohn or our alkaline sucrose gradient technique in slow reorienting zonal rotors to define the DNA damage and repair kinetics after treatment with radiation, drugs, or combinations of drugs with radiation in tumors and normal cells isolated from or near the tumors. The tumor model will be either intracerebral or subcutaneous 9L tumors in Fisher 344 rats. Normal cells will be (1) cerebellar neurons or (2) lymphocytes and macrophages isolated by centrifugal elutriation from 9L tumors. The alkaline elution analysis will determine the relative amount of DNA-DNA crosslinks, DNA-protein crosslinks and DNA strand breaks in treated cells. Repair kinetics will be evaluated by the relative rates and extent of removal of these different types of damage. Differences in the DNA repair kinetics among the various cells studied may be due to differences in the DNA template structure or differences in the availability or function of the repair enzymes. The susceptibility of chromatin isolated at various steps of the repair process to digestion by nucleases or its ability to support polymerase activity will be used to evaluate the template theory. Assays for Alpha and Beta polymerase activity from both normal and malignant cells will be used to test the repair enzyme theory. We will attempt to determine whether critical periods in the DNA repair process are associated with changes in either the DNA template structure or the quantity or quality of the repair enzymes.