In recent years, much effort has been directed to understanding mechanisms of clinical anticancer drug resistance. Markers of multidrug resistance (MDR) - overexpression of the mdr1 gene and its product, P- glycoprotein (Pgp) - have been shown to be associated with some clinically resistant tumors. However, only incremental progress has bee made in assessing the role of MDR in clinical drug resistance, likely du in part to the fact that not all drug resistant tumors express Pgp. Indeed, other forms of MDR have been described experimentally that may complicate assays for MDR in clinical specimens. A long-term goal of th proposed studies is the development of microdetection and in situ assays to detect MDR, regardless of the mechanism, in the tumor cells of patients. Our approach has been to characterize biochemical and molecular features of MDR cells and to develop single-cell functional an molecular assays based on these resistance-associated features in order to permit detection of such drug resistant cells in patients' tumors. In this way, we discovered mutations in the topo IIalpha gene associated with an altered topoisomerase II-form of MDR (at MDR) and exploited this finding in a molecular assay (single-strand conformational polymorphism; SSCP) that permitted screening of a large number of cell lines and leukemic specimens for these and other mutations. We are developing single-cell functional assays, based on resistance-associated features, to detect MDR in clinical specimens. In this grant-period, I propose to test the hypotheses that [1] single-cell functional assays for drug resistance can predict the situation in patient's tumors and [2] it is possible to directly image functional drug resistance in tumors in situ. The following specific aims are designed to test these hypotheses: (1) determine in PCR-based and immunologic assays the association of resistance-associated proteins with MDR; (2) develop fluorescence digital image microscopy (FDIM) to quantitate and immunolocalize resistance-associated proteins and to quantitate DNA damage (comet assay and apoptosis in single-cells; (3) develop single photon emission computed tomography (SPECT) methods for functional analysis of Pgp in solid tumors in situ; and (4) apply these findings and assays (SSCP, quantitate PCR, FDIM, comet, SPECT) to patients' tumors.
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