INCREASING THE THERAPEUTIC EFFICACY OF MANY ANTICANCER DRUGS.

INCREASING THE THERAPEUTIC EFFICACY OF MANY ANTICANCER DRUGS.THE LONG-RANGE GOAL OF THIS PROPOSAL IS TO DEVELOP AN IN VITRO TEST TO IMPROVE SELECTIONS OF THERAPIES FOR INDIVIDUAL BREAST CANCER PATIENTS BY PREDICTING RESPONSE TO METHOTREXATE. EXISTING ASSAYS FOR BREAST CANCER CELLS CANNOT DETERMINE METHOTREXATE SENSITIVITY BECAUSE OF REQUIREMENTS FOR FEEDER LAYERS AND MEDIUM COMPONENTS WHICH CAN RESCUE SENSITIVE CELLS. THIS PROPOSAL WILL EVALUATE TWO DIFFERENT APPROACHES: 1.IMPROVE AN EXISTING CLONOGENIC ASSAY BY MANIPULATING A SERUM-FREE MEDIUM BASED UPON MCDB 170 SO THAT METHOTREXATE TOXICITY IS ACHIEVED, AND 2.DEVELOP A TOTALLY DIFFERENT ASSAY FOR METHOTREXATE RESISTANCE WHICH MEASURES UPTAKE OF FLUORESCEIN-CONJUGATED DRUG USING A FLUORESCENCE ACTIVATED CELL SORTER (FACS). THE LATTER ASSAY DOES NOT REQUIRE CLONAL GROWTH. PHASE I WILL ESTABLISH THE NECESSARY MEDIUM FORMULATION FOR THE CLONAL ASSAY AND SHOW THAT METHOTREXATE DOSE RESPONSE CURVES ARE FEASIBLE FOR NORMAL AND TUMOR-DERIVED EPITHELIAL CELLS. ADDITIONALLY, PHASE I WILL CONTINUE THE FACS STUDIES AND EXPAND POTENTIAL SOURCES OF CLINICAL MATERIAL AND CORRELATIONS SO THAT DURING PHASE II BOTH ASSAYS MAY BE CONTINUED AND EVALUATED FOR EFFICACY AND PRACTICALITY. THE ASSAYS DEVELOPED IN PHASE I WILL PROVIDE A MEANS TO ISOLATE METHOTREXATE-RESISTANT CELLS WHOSE MECHANISMS OF RESISTANCE WILL BE EXAMINED DURING PHASE II. THUS, THESE IN VITRO ASSAYS WILL NOT ONLY BE USEFUL FOR PREDICTIVE PATIENT SENSITIVITY TESTING, BUT SHOULD ALSO PROVIDE A BASIS FOR IMPROVED DRUG DESIGN.