The long-term objectives of this proposal are: 1) to accurately quantify tumor hypoxia with the ultimate goal of identifying patients likely to benefit from therapies directed against hypoxic cells, and 2) to understand the physiological and biochemical factors that contribute to the expression, development, and maintenance of hypoxic cells in solid tumors. There are 3 specific aims. First, the binding patterns of the hypoxia markers pimonidazole and NITP in SCCVII murine tumors and SiHa xenografts will be interpreted by simultaneous application of marker binding measured by flow cytometry with hypoxic fraction measured by the comet assay. This should provide a rational way to determine the meaningful radiobiological hypoxic fraction from flow histograms of maker binding in human tumor cells and from fine needle aspirates measured using quantitative fluorescence microscopy. In the second aim, the capacity of murine and human tumor cells to repair DNA damage in vivo and the fidelity of that repair will be measured. The hypothesis is that chronically hypoxic (nutrient depleted) tumor cells repair DNA damage more slowly and less accurately than well-oxygenated cells. DNA strand breaks and base damage will be measured using the alkaline comet assay in cells recovered from SCCVII, RIF-1, U87, and SiHa tumors at different times after irradiation or treatment with the hypoxic cytotoxin, tirapazamine. Tumor cells sorted from hypoxic and oxic areas will be examined for possible differences in repair kinetics. To examine the fidelity of repair, mutation at the HPRT locus will be measured in RIF-1 and U87 tumor cells recovered after irradiation in vivo, and expression in vivo or in vitro. Finally, """"""""reporter genes"""""""" will be exploited as an aid to research on tumor hypoxia and evaluation of hypoxic cell response to treatment. SiHa human cervical tumor cells will be transfected or transduced with constructs expressing green fluorescent protein (GFP) under the control of a hypoxia response element, HIF-1alpha. New short-lived GFP, as well as proteins that fluoresce at different wavelengths, will be used to develop more versatile models. To validate the models, expression of GFP will be analyzed in relation to proliferation, hypoxia marker binding, and clonogenicity following irradiation.
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