Although multidrug resistance (MDR) is well-documented, its clinical relevance remains to be established. Accordingly, efforts to characterize MDR in human tumor cells are warranted. While P- glycoprotein (Pgp) clearly plays a role in mediating MDR, other forms of natural product MDR exist. One form is restricted to drugs that interact with DNA topoisomerase (topo) II and is associated with alterations in this enzyme (at-MDR). Much remains to be learned about both forms of MDR. For example, anticancer drug binding sites in Pgp and in the DNA- topo complex are unknown. Further, the mechanisms by which expression of Pgp and topo II are regulated in MDR cells remain to be uncovered. There are two forms of topo II, topo alpha (170 kd) and topo beta (180 kd), but their relative contributions to at-MDR are unclear and their regulation is poorly understood. Finally, it can be asked whether resistance to anti-topo drugs might be due in part to an inability of the cell to mount certain stress responses. Accordingly, the long-range goal of the proposed research is to characterize both Pgp-MDR and at-MDR at the cellular, biochemical, and molecular level. To achieve this goal, I propose to test the following hypotheses: (1) there are specific drug-binding sites in Pgp and in binary complex of DNA and topoisomerase II that are critical for drug action and are altered in MDR; (2) topo alpha and beta are regulated differently in at-MDR cells than in drug- sensitive cells; (3) topo beta is important in at-MDR; and (4) a component of at-MDR involves the inability of the cell to mount certain stress responses that normally lead to induction of new proteins and alterations in DNA repair. To test these hypotheses, the following specific aims are proposed: (1) compare the drug and modulator binding sites on P-glycoprotein by photoaffinity labeling, peptide sequence analysis, and mutagenesis studies; (2) determine the regulation of Pgp by rugs and modulators through study of their effects on mdrl/Pgp regulation at the transcriptional and post-transcriptional levels; (3) compare the role of topo II with that of topo II alpha in at-MDR by examining the effects of drugs on the activities and subcellular distribution of these isoforms; (4) assess the transcriptional and post- transcriptional regulation of both topo alpha and topo beta in drug- sensitive and at-MDR cells; (5) determine by photoaffinity and sequencing methods that anti-topo II drug binding sites in the binary complex of topo II and DNA in drug-sensitive and -resistant cells; and (6) determine whether certain stress responses, DNA damage-inducible genes, or gene-specific DNA repair are important in at-MDR.
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