PROBES FOR FLOW CYTOMETRY

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The long term objective of this project is the development of new probes, methods and instrumentation for analysis of individual cells by flow or image cytometry, applicable for tumor diagnosis and/or prognosis; the developed methodologies will be applied In studIes of the cell cycle and cell sensitivity to antitumor drugs. The specific aims are focused on development of markers of apoptosis, and of cell proliferation. Development of the method based on DNA in situ strand break labeling that employs exogenous terminal transferase will be continued; the technique will be combined with analysis of cell proliferation. Cell proliferative potential will be estimated by both, the analysis of expression of the cell cycle specific proteins, cyclin E and B1 and measurement of DNA replication. The latter will be detected by a novel approach, which combines photolysis of the newly replicated DNA with the detection of DNA strand breaks. This approach, which offers several advantages over the conventional detection of DNA replication by 5-bromodeoxyuridine antibody, will also be tested with respect to its ability to measure DNA repair. A combination of fluorochromes will be selected that will also allow measurement of cellular DNA content. The multivariate analysis of DNA content, cell death (apoptosis) and proliferation (cyclin E and B1 expression or DNA replication) will be applied in studies of human leukemias, before and during the course of chemotherapy, and in breast cancer; the prognostic value of the parameters measured will be evaluated. it is expected that such a combined analysis, especially involving estimates of the G1 and G2 cyclins, will be of high prognostic value, and will be used during treatment, to monitor tumor response.