The long term objective of this application is to develop methods that enable the rapid identification of the mechanistic type and frequency of antineoplastic drug resistant cells within populations of blast cells obtained from patients with acute nonlymphocytic leukemia (ANLL). The importance of such methodologies cannot be understated, since they will not only aid in the immediate selection of therapy, but will also give direction to future research in the design of new therapeutic approaches, aimed at overcoming those mechanisms of drug resistance identified. These studies will focus on the two most effective agents currently available for treatment of ANLL, daunorubicin (DNR) and 1-B-D-arabinofuranosylcytosine (ara-C). We will utilize state-of-the-art flow cytometric and cell sorting technology to enhance detection of drug resistant cells. Our preliminary studies reveal that our flow cytometer can accurately detect sub-populations as infrequent as 0.016% of the total population.
Our specific aims propose: A. to refine methods of flow cytometric evaluation of intracellular DNR and to investigate the relationship between intracellular DNR content or concentration as determined by flow cytometry and neoplastic cellular sensitivity to the drug; B. to establish optimal conditions of flow cytometric detection of ara-C incorporation into the DNA of single cells, utilizing a fluorescent antibody technique. To aid in achieving this end, we will attempt to develop a monoclonal antibody which recognizes ara-C incorporated into DNA. DNA synthesis during ara-C exposure will be quanitified in individial cells by utilizing the established flow cytometric method for measuring bromodeoxyuridine incorporation into DNA; C. to correlate the type and frequency of biological or biochemical alterations observed in blast cells obtained from patient with ANLL with clinical response of these patients to ara-C and DNR.
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