MICRODETECTION ASSAY FOR DRUG-RESISTANT TUMORS

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Understanding multiple drug resistance (MDR) has obvious clinical importance. MDR is typified by the overabundance of a "marker" protein, known as "P-glycoprotein" or gp180", and its appearance can be traced to amplification and/or overexpression of the gene coding for it. Some tumor samples from patients have been shown by blotting assays to have this protein and overexpress its mRNA. However, gp180 is not invariably a marker of MDR. Recent work from this laboratory has identified "atypical" MDR (at-MDR) in human leukemic cells that are resistant and cross-resistant to anthracyclines and epipodophyllotoxins, but are sensitive to vinca alkaloids. This at-MDR is of potential clinical importance, since many treatment protocols involve combinations of these classes of drugs. At-MDR cells do not overexpress the MDR marker protein, or its mRNA; hence, the available antibody and cDNA probes for "classic" MDR will fail to detect this form of MDR in clinical specimens. A specific aim of this proposal is to develop monoclonal antibody and cDNA probes that will distinguish at- MDR cells from their drug-sensitive counterparts and will be suitable reagents in a clinical microdetection assay. To improve detection of MDR in the face of tumor cell heterogeneity and antigen modulation, we intend to develop single cell micro- detection assays for MDR- and at-MDR-associated proteins and mRNAs, using immunohistochemical and in situ hybridization methodologies. Such assays will be applied to a variety of tumor specimens and normal tissues, and the results correlated with clinical disease features and treatment outcome. Finally, the MDR marker protein, gp180, may have different functional domains associated with drug binding, cell growth and cytotoxicity. The hypothesis will be tested directly by developing panels of antibodies that will identify these sites. Such antibodies may be useful in a microdetection assay and potentially could have therapeutic benefit. In summary, the long term-goal of the proposed work is the development of a reliable clinical microdetection assay for the presence of MDR and at-MDR that will ultimately permit the development of more specific therapy. To do so, it will be necessary to develop antibody and nucleic acid reagents that will identify at-MDR, since existing reagents will only detect "classic" MDR. These reagents will be used to develop a panel of sensitive and specific clinically useful single- cell and blotting assays, as determined is prospective and retrospective studies.