This multifaceted research proposal focuses on the molecular pharmacology of etoposide (VP-16) and the modulation of VP-16 activity in sensitive human leukemia cells in vitro and in cells selected for resistance in the presence of VP-16 or vinblastine (VLB). The long term goals of this project are to: 1) define the mechanisms which regulate cell disposition and cytotoxicity of VP-16 in sensitive and resistant cells; 2) characterize the development of VP-16 resistance by distinguishing membrane transport alterations from changes in levels and activities of DNA Topoisomerase II (Topo II) or modulators of Topo II; 3) develop strategies for clinical combination chemotherapy (involving VP-16) to overcome resistance. VP-16/microtubule inhibitor combination studies will focus on the modulation of VP-16 binding affinities to tubulins, an outgrowth of results from this laboratory demonstrating Vinca alkaloid promotion of VP-16 binding to tubulin. These drug combinations will be used to assess cytotoxicity in: 1) VP-16 resistant, Vinca alkaloid sensitive K562 cells which have altered topo II activity; 2) VLB transport resistant, VP-16 cross-resistant CCRF-CEM cells. Synergistic cytotoxicity of VP-16/VLB combinations in VP-16 resistant cells suggests that enhanced VP-16 tubulin binding may play a cytotoxic role when topo II activity is altered. The presence and role of cell surface tubulin will be investigated in the context of VP-16/microtubule inhibitor combinations especially in VLB transport resistant CEM cells which overexpress p-glycoprotein. In separate studies, topo II in VP-16 resistant cells will be characterized in terms of: 1) drug effects on topo II binding to and unknotting/unwinding DNA; 2) the role of nucleotides (mono- , di- , tri- , nonhydrolyzable) in drug-induced DNA damage; 3) reversal of drug/enzyme binding and damage to DNA; 4) collateral sensitivity of alkylating agents in VP-16 resistant cells; 5) sequence specificity for VP-16 induced DNA cleavage; 6) VP-16 effects on transcription and replication. Results of the proposed studies will further understanding of VP-16 activity when used alone or in combination and will contribute fundamental information of gene regulatory systems and mechanisms of acquired drug resistance.
