The basic theme of this proposal is to study the effect of calmodulin inhibitors on cellular targets governing a cytotoxic response to adriamycin and cross-resistant drugs in progressively adriamycin-resistant tumor models. The tumor models to be characterized will include the parent-sensitive and progressively adriamycin-resistant variants of L1210 mouse leukemia, B16-BL6 mouse melanoma and HL-60 human promyelocytic leukemia. The calmodulin inhibitors to be evaluated are trifluoperazine, and 1,3-dihydro-1-[1-[(4-methyl-4H, 6H-pyrrolo[1,2-a][4,1]-benzoxazepin-4- yl)methyl]-4-piperidinyl]-2H-benzimidazol-2-one(l:l)maleate (CGS9343B) a selective calmodulin inhibitor which does not inhibit protein kinase activity. The sensitive and progressively adriamycin-resistant tumors will be characterized to determine: (a) the cellular accumulation/retention and subcellular distribution of calmodulin inhibitors in the absence or presence of adriamycin, etoposide and vincristine using radiolabelled drugs and/or high performance liquid chromatography (HPLC); and (b) the role of extranuclear factors in governing adriamycin or etoposide induced DNA damage and repair/rejoining of these lesions in the absence or presence of calmodulin Inhibitors using isolated nuclei alone or nuclei reconstituted with membrane and/or cytosol fractions, by the technique of alkaline elution. Further, adriamycin or etoposide induced DNA damage based on Interaction with the 170 kDa and 180 kDa isoforms of topoisomerase II will be determined by band depletion-immunoblotting technique. Alterations in phosphorylation of P-glycoprotein and topoisomerase II and its modulation by calmodulin inhibitors will be determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates. The phosphorylated immunoprecipitates of P-glycoprotein and topoisomerase II will be further characterized to determine phosphorylated amino acids by thin layer electrophoresis and phosphopeptides following digestion with Staphylococcus aureus V8 protease. Purified topoisomerase II will be isolated from sensitive and adriamycin-resistant cells by fast protein liquid chromatography and characterized for catalytic activity based on P4 unknotting, drug induced DNA protein cross linking using uniquely 3' end labelled SV-40 DNA and phosphorylation by calcium/calmodulin dependent protein kinase 11. The role of calmodulin inhibitors in modulation of resistance and emergence of a resistant phenotype will be characterized in tumor cells selected for resistance to a combination of adriamycin plus, trifluoperazine, by evaluating cytotoxic response, induction of DNA damage and phosphorylation of putative targets viz. P-glycoprotein and topoisomerase II, in the absence or presence of calmodulin inhibitors. The proposed studies from a pharmacological and biochemical standpoint should in the long term help us understand the targets involved in the acquisition and expression of resistance to adriamycin and other inhibitors of topoisomerase II, or vinca alkaloids and the potential role of calmodulin inhibitors in modulating cellular effects governing cytotoxicity.
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