This proposal is designed to enhance the current understanding of the interaction between DNA topoisomerase I and inhibitory antineoplastic drugs. Knowledge of factors involved in the interaction between the enzyme and inhibitors such as camptothecin would be of benefit in the clinical application of these agents. The studies proposed in Aim l are an extension of prior work in which a novel mechanism of cellular resistance to camptothecin was identified, involving a point mutation at position 361 in topoisomerase I. Preliminary studies suggest that this mutation confers resistance to the effects of camptothecin on the enzyme, and that additional mutations in this region impair the DNA cleavage step of the topo 1 catalytic cycle. These findings will be confirmed and extended by the use of oligonucleotide cleavage assays and gelshift assays performed with bacterially expressed mutant proteins. Additional studies will evaluate the effects of these mutations on camptothecin binding and on the inhibitory activity of noncamptothecin topoisomerase T poisons such as Hoechst dye 33342 and actinomycin D. Since the cellular response to topoisomerase I inhibition may be influenced by proteins associating with the enzyme, Specific Aim 2 is designed to identify such proteins. Preliminary work using affinity chromatography with an immobilized topoisomerase I fusion protein has identified several putative binding proteins, and microsequencing has identified one of these proteins as nucleolin. Studies in this Aim are designed to confirm this finding and to localize the site of binding to topoisomerase I by the use of deletion mutants. Additional studies will attempt to identify other binding proteins and to investigate the effects of these proteins on topoisomerase I function and on the interaction between topoisomerase I and inhibitors such as camptothecin.
| Hait, William N; Rubin, Eric; Goodin, Susan (2005) Tubulin-targeting agents. Cancer Chemother Biol Response Modif 22:35-59 |
| Hait, William N; Rubin, Eric; Goodin, Susan (2003) Tubulin-targeting agents. Cancer Chemother Biol Response Modif 21:41-67 |
| Hait, William N; Rubin, Eric; Goodin, Susan (2002) Tubulin-targeting agents. Cancer Chemother Biol Response Modif 20:71-97 |
| Hait, W N; Rubin, E; Goodin, S (2001) Tubulin targeting agents. Cancer Chemother Biol Response Modif 19:59-83 |
| Saleem, A; Edwards, T K; Rasheed, Z et al. (2000) Mechanisms of resistance to camptothecins. Ann N Y Acad Sci 922:46-55 |
| Edwards, T K; Saleem, A; Shaman, J A et al. (2000) Role for nucleolin/Nsr1 in the cellular localization of topoisomerase I. J Biol Chem 275:36181-8 |
| Pond, C D; Li, X G; Rubin, E H et al. (1999) Effects of mutations in the F361 to R364 region of topoisomerase I (Topo I), in the presence and absence of 9-aminocamptothecin, on the Topo I-DNA interaction. Anticancer Drugs 10:647-53 |
| Haluska Jr, P; Saleem, A; Rasheed, Z et al. (1999) Interaction between human topoisomerase I and a novel RING finger/arginine-serine protein. Nucleic Acids Res 27:2538-44 |
| Haluska Jr, P; Rubin, E H (1998) A role for the amino terminus of human topoisomerase I. Adv Enzyme Regul 38:253-62 |
| Haluska Jr, P; Saleem, A; Edwards, T K et al. (1998) Interaction between the N-terminus of human topoisomerase I and SV40 large T antigen. Nucleic Acids Res 26:1841-7 |
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