The overall aim of this project is to improve human cancer therapy by developing and applying techniques to rapidly assess the efficacy of therapeutic agents, to detect resistant tumor cells at low frequency and to determine the cytokinetic characteristics of tumors before and during therapy. These techniques will be developed first in model murine tumors and then extended to human leukemias. We propose to complete development of our flow cytometric assay for cellular DNA content and amount of bromodeoxyuridine (BrdUrd/DNA assay). This will include production and characterization of new high affinity antibodies against BrdUrd incorporated in DNA for use as immunochemical reagents. We will apply this assay to assess the sensitivity of tumor cells to therapeutic agents that inhibit cell proliferation (eg. cytosine arabinoside) by measuring the extent to which these agents surpress DNA synthesis in cells with S-phase DNA content grown in vitro or in vivo. We also propose to detect ara-C killed cells directly by measuring ara-C induced DNA damage. We will develop monoclonal antibodies against ara-C incorporated in DNA and use these for immunofluorescence staining. The immunofluorescence per cell will be measured flow cytometrically along with total DNA content (ara-C/DNA assay). We will apply the ara-C/DNA and BrdUrd/DNA assays and high speed cell sorting to detect rare ara-C resistant cells that might eventually lead to therapy failure. We propose to apply our BrdUrd/DNA assay to analysis of the cytokinetic status of murine and human tumors before and during therapy. Cytokinetic information will be determined by measuring the rate of cell cycle traverse of cells labeled in vitro or in vivo with BrdUrd.
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