The basic theme of this application is to establish mechanisms by which the topoisomerase II poisons adriamycin or etoposide and the inhibitors of calcium-calmodulin regulated processes, trifuluoperazine and 1-[N-O- bis(1,5-isoquinolinesulfony)-N-methyl-L-tyrosyl-4phenylpiperazine(KN- 62) co-operate in sensitizing resistant cells. We hypothesize that intracellular free calcium and the phosphorylation of topoisomerase II are required for etoposide induced DNA cleavable complex formation and cytotoxicity. Experiments will be carried out using sensitive or progressively adriamycin-resistant HL-60 and L1210 tumor model systems. To test whether the sensitizing effect of calmodulin inhibitors on etoposide or adriamycin is dependent on free calcium, cells wil be treated with an intracellular calcium buffer and drug accumulation, DNA cleavable complex formation and apoptosis will be determined as potential mechanisms involved in enhancing cytotoxicity. The effect of calmodulininhibitors on pohosphorylation of topoisomerasde II will be evaluated in control or calcium bufffer treated cells labelled with [32P]-orthophosphoric acid. Using topoisomerase II immunoprecipitates we will characterize, drug induced DNA cleavable complex formation by band depletion and phosphopeptides by two dimensional tryptic mapping. Stability of drug induced DNA damage and its dedpendence on the phosphorylation of topoisomerase II will also be determined. The cell cycle phase specificity of calmodulin inhibitors will be evaluated in synchronized cell populations by determining: (a) drug stablilized DNA cleavable complex formation, apoptosis and cytotoxicity; and (b) the phosphorylation of topoisomerase II and its peptides in immunoprecipitates. In the long term a ciochemical and pharmacological characteriszation of events regulating the modulation of cytotoxicity by calmodulin inhibitors should aid in the understanding of mechanisms of resistance to clinically usefule topoisomerase II inhibitors.
Showing the most recent 10 out of 35 publications
