The objective of this project is to characterize the clinical mechanism(s) of resistance to the anti-cancer agents etoposide (VP-16) and to implement a strategy to overcome resistance. The applicant hypothesizes that acquired resistance to VP-16 in human K562 leukemia cells is due in part to hypophosphorylation of DNA topoisomerase II alpha (topo II). The major goal is to elucidate the protein kinase signalling pathway responsible for topo II hypophosphorylation and to establish that posttranslational modification of this enzyme target is seminal to clinical VP-16 resistance. Bryostatin 1, a protein kinase C (PKC) activator, will be examined for its ability to translocate/activate nuclear PKC and subsequently upregulate topo II phosphorylation leading to increased VP-16-induced cytotoxicity in resistant K562 cells and in marrow blasts from leukemia patients who have relapsed after receiving therapy with topo II inhibitors. In preliminary results using bryostatin 1, nuclear translocation/activation of beta II PKC has been implicated in the regulation of topo II phosphorylation in resistant K562 cells. In addition, bryostatin 1 potentiates VP-16 cytotoxicity in resistant K562 cells using a clonogenic assay. Therefore, the specific aims are: 1. To further establish the role of beta II PKC and/or other protein kinases in phosphorylation of topo II in resistant K562 cells and to demonstrate that bryostatin 1 stimulates topo II phosphorylation through a beta II PKC signalling pathway; 2. To determine the ability of bryostatin 1 to upregulate topo II phosphorylation, potentiate VP-16-induced topo II/DNA complexes and increase VP-16-induced cytotoxicity in freshly obtained marrow blasts from relapse leukemia patients. Results obtained will better define posttranslational modifications of topo II responsible for VP-16 resistance and may translate to use of VP-16/bryostatin 1 combinations as an effective clinical strategy to treat drug-resistant malignancies.
