A major problem in the treatment of acute leukemia is the development of resistance to chemotherapy and subsequent relapse. Ultimately, patients may fail to respond to any therapy, and exhibit clinical resistance not only to many drugs, but also to steroids. Despite the appearance of multiple-drug resistance in these patients, little is known about its biochemical expressions. Although biochemical lesions associated with multiple drug resistance have been described in animal tumor models. it is not presently known if cells taken directly from patients who are clinically refractory to many drugs actually display the multiple drug- or """"""""pleiotropic""""""""-resistance phenotype, characterized by the presence (or absence) of certain marker proteins; this appears to be equally true for steroid resistance, which has likewise been shown in animal cells to be associated with certain marker proteins. Identification of these marker proteins for multiple drug- or steroid-resistance in patient-derived tumor cells could provide a basis for a drug- or steroid-resistance in patient-derived tumor cells could provide a basis for a microdetection assay that might predict clinical drug resistance. Accordingly, the primary objective of this grant proposal is to develop a microdetection assay for the presence of small numbers of drug-resistant cells bearing these markers in heterogeneous populations containing primarily sensitive cells. The proposed assay will differ from its predecessors in that it will be based solely on phenotypic differences between drug-resistant and -sensitive cells. This assay will be independent of cell growth, cell culture, """"""""target"""""""" enzyme activity and the uptake or incorporation of drugs and precursors of macromolecules. The ability to detect such cells reliably will require a rigorous biochemical characterization of resistance-associated molecular changes as determined in part by high-resolution analytical two-dimensional gel electrophoresis, a specific aim of this proposal. Antibodies developed against such marker molecules (another specific aim) will be used in the detection assay, which will be based on immunofluorescent technics employing flow cytometry, as well as on radioimmunoassay (other aims). The long-term goals of such proposed research are to (i) determine whether multiple drug-resistant cells exist in patient tumor cells, (ii) determine if they can be detected reliably, and (iii) develop a battery of tests based on resistance markers that may have ultimate clinical utility by facilitating the individualization of therapy.
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