The objective of this project is to characterize the mechanism(s) of resistance to the bisdioxopiperazine class of DNA topoisomerase II (topo II) catalytic inhibitors. Compared to parental CHO cells, a subline (DZR) with acquired resistance to dexrazoxane (ICRF-187) exhibits no change in topo II catalytic activity but shows a dramatic decrease in the ability of ICRF-187 to: 1) interfere with initial formation of VP-16-induced topo II-DNA complexes; 2) """"""""chase-out"""""""" preformed VP-16- induced topo II-DNA complexes. We hypothesize that the stable acquired resistance phenotype to ICRF-187 in DZR is due to changes in topo II structure/function that alters its interactions with DNA and/or drugs.
Specific Aims are to: 1. determine the molecular basis for DZR cells resistance to ICRF-187 by sequencing of topo II cDNA and to demonstrate further the biochemical mechanism(s) of bisdioxopiperazine resistance. 2. synthesize and utilize a photoactivatible VP-16 analog for the study of ICRF-187 mechanisms of action and resistance. 3. test the hypothesis that ICRF-187 inhibition of topo II will upregulate DNA topoisomerase I levels and sensitize cells to inhibitors such as topotecan. Results obtained will establish more precisely the mechanism of action of drugs that inhibit topo II without inducing topo II-DNA cleavable complexes and will lead to strategies for clinical use of these agents alone or in combination with other topoismerase inhibitors.
