RADIOBIOLOGICAL TECHNIQUES IN HUMAN CANCER THERAPY

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.