The presence of a significant percentage of hypoxic cells (10-25%) in animal and human tumors can influence the outcome of both radiotherapy and chemotherapy. Although the hypoxic fraction in animal tumors often increases with size, no tumor characteristic such as histology, size volume doubling time, site of growth, or degree of differentiation has been found to predict either the presence of hypoxic cells or the hypoxic fraction in human tumors. Thus, a rapid, accurate method for detecting and quantitating hypoxic cells in human tumors could make a substantial impact on the treatment of cancer by allowing both radiotherapy and chemotherapy to be individualize based on he characteristics of each patient's tumor. There are two classes of radiation-induced DNA damage whose formation exhibits an oxygen dependency similar to that for radiation-induced cell killing. The O2 dependency for formation of DNA-protein crosslinks (DPCs) is virtually the mirror image of the O2 dependency for radiation-induced cell kill. The formation of radiation-induced DNA strand breaks (SBs) has an O2 dependency virtually identical to the O2 dependency for radiation- induced cell kill. Both phenomena lead to a retardation in the removal of DNA from the filter during alkaline elution. After a given volume of elution, the amount DNA remaining on the filter is linearly related to the percentage of hypoxic cells in the irradiated tissue. The goals of the proposed research are to: 1) determine if an alkaline elution assay for SBs and DCPCs can reliably measure the hypoxic fraction of mouse and human tumors, 2) provide the data to properly select the radiation dose and sampling procedures for animal and human trials using this assays, and 3) characterize the chemical nature and location of the DPCs measured by this DNA assay as a function of the O2 concentration, metabolic state, proliferative state and time between radiation exposures. Three mouse tumors (RIF, SCCVII, KHT) and 3 human colon tumors (clone A, HCT-15, WiDR) grown as xenografts in nude mice will be used to achieve these objectives. The chemical nature of the DPCs located near the nuclear matrix or in the chromatin will be determined using GC/MSD techniques. If, 1) induction of SBs and DPCs is minimally influenced by biological and physiological factors other than the O2 concentration near the chromatin, and 2) the assay reliably predicts the hypoxic fraction of experimental mouse and human tumors, studies will be started in the clinic to test its usefulness in predicting the radiation responded of head and neck tumors or cervical carcinoma, and in the lab to validate noninvasive techniques for detecting and quantitating hypoxic cells in tumors and normal tissue.
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