The basic theme of this proposal is to study the effect of calmodulin inhibitors in vitro and in vivo in modulating the cytotoxic response to adriamycin and cross-resistant drugs in progressively adriamycin-resistant tumor models. The tumor models to be studied will include the parent-sensitive and progressively adriamycin-resistant variants of the P388 and L1210 mouse leukemia and B16-BL6 mouse melanoma. The calmodulin inhibitors to be evaluated are trifluoperazine, and N-(4-aminobutyl)-5-chloro-2- naphthalenesulfonamide (W-13) a significantly less lipophilic and """"""""more specific"""""""" inhibitor of calmodulin. The sensitive and progressively resistant tumors will be characterized to determine the effect of calmodulin inhibitors on: (a) distribution of adriamycin in sub-cellular fractions obtained from cell homogenates by differential centrifugation and quantification of adriamycin levels by high performance liquid chromatography (HPLC); and (b) adriamycin induced DNA protein cross links, DNA single and double strand breaks, and the repair/rejoining of these lesions using the technique of alkaline and neutral elution. The effects of caffeine on adriamycin cytotoxicity in the absence and presence of calmodulin inhibitors will be focused on determining alterations in adriamycin accumulation, changes in cellular free calcium concentrations, damage to DNA and cytotoxicity. Cellular adrlamycin levels wlll be measured by HPLC and also by laser flow cytometry. Cellular free calcium levels will be determined with the fluorescent calcium indicator Quin-2, and cytotoxicity by soft- agar colony assay. The relationship between progressive adriamycin resistance, cross resistance characteristics, and the modulating effect of calmodulin inhibitors with antltumor agents such as vinca alkaloids, epipodophyllotoxins, anthracycline and non-anthracycline agents with variable affinity to bind to DNA will be evaluated in vivo. Antitumor effects in vivo will be determined bv measuring changes in prolongation of life span with P388 and L1210 leukemia, and changes in tumor volume and/or reduction of pulmonary metastases with B16-BL6 melanoma. Acquisition of resistance to adrlamycin in vivo will be evaluated with the B16-BL6 melanoma. Metastatic characteristics of resistant cells will be based on formation of experimental and spontaneous metastases, and the response to antltumor effects of adriamycin in vltro and in vivo will be determined by soft-agar colony assay and reductlon in tumor burden respectively. The proposed studies should in the long term help us understand the molecular basis for the acquisition and expression of resistance to adriamycin and the potential role of calmodulin inhibitors in modulating adriamycin cytotoxicity.
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