The long-term objectives of this proposal are 1) to develop the comet assay as a method to quantify radiobiologically hypoxic cells in human tumors with the ultimate goal of identifying patients likely to benefit from therapies directed towards these resistant cells, and 2) to understand the physiological and biochemical factors which contribute to the development, maintenance, and expression of hypoxia in solid tumors. The specific goals for the next 3 year period are to: 1) develop a method in murine tumors with potential clinical applicability for the measurement of perfusion-limited hypoxia. 2) examine the response of human tumor fine needle aspirates in the comet assay in terms of heterogeneity between aspirates, change in hypoxic fraction following palliative therapy, and ability to separate the response of tumor from normal cells. 3) compare the DNA damaging ability of PD 144872 in murine tumor and normal tissues with chemically related drugs RSU 1069 and RB 6145. 4) determine whether hypoxic fraction for rodent tumors measured using the comet assay and hypoxic fraction measured using the paired survival curve method correlate for single doses and after a fractionated treatment regime, and whether the same relationship holds across tumor lines. The comet assay has recently been developed to detect radiobiologically hypoxic cells in murine and human solid tumors; applying and improving this single cell gel electrophoresis method forms a focus for this renewal application. Radiobiologic hypoxic fraction will be measured in murine tumors (SCCVII murine tumors and SiHa human cervical xenografts) before and following fractionated radiation treatments (5 4 Gy; 2 20 Gy), and results with the comet assay will be compared with a standard clonogenicity assay performed using the same cell suspension. Cells will be obtained by multiple fine needle aspiration biopsy of human tumors (primarily breast cancer recurrences) immediately following the first and last dose of a palliative treatment (5 4 Gy); heterogeneity in hypoxic fraction and DNA content, and change in hypoxic fraction following treatment will be assessed. In addition, a method will be developed for the detection of perfusion- limited hypoxia in murine tumors which may eventually be applied in the clinic. This method will employ BrdUrd and IdUrd incorporated into S phase tumor cells and detected by fluorescence-tagged antibodies using flow or static cytometry; areas containing cells which incorporate one but not both of these drugs will be characterized as undergoing transient changes in blood flow.