Nucleolin functions in ribosome biogenesis and contains an acidic N terminus that binds nuclear localization sequences. In previous work we showed that human nucleolin associates with the N-terminal region of human topoisomerase I (Top1). We have now mapped the topoisomerase I interaction domain of nucleolin to the N-terminal 225 amino acids. We also show that the Saccharomyces cerevisiae nucleolin ortholog, Nsr1p, physically interacts with yeast topoisomerase I, yTop1p. Studies of isogenic NSR1 ؉ and ⌬nsr1 strains indicate that NSR1 is important in determining the cellular localization of yTop1p. Moreover, deletion of NSR1 reduces sensitivity to camptothecin, an antineoplastic topoisomerase I inhibitor. By contrast, ⌬nsr1 cells are hypersensitive to the topoisomerase II-targeting drug amsacrine. These findings indicate that nucleolin/Nsr1 is involved in the cellular localization of Top1 and that this localization may be important in determining sensitivity to drugs that target topoisomerases. Human Top11 is a nuclear protein involved in the regulation of DNA structure and is the target of an important new class of antineoplastic drugs, the camptothecins (1, 2). Studies in yeast provide convincing evidence that Top1 is the sole cellular target for camptothecins, with this knowledge facilitating attempts to understand the mechanisms by which the drug destroys cells (3-5). Top1 is a monomeric protein that relaxes supercoiled DNA by creating a transient single-strand nick, with this nick involving the covalent attachment of Top1 to the DNA phosphate backbone via a phosphotyrosine bond. Recent structural studies of Top1 and the Top1-DNA covalent complex led to molecular models of the mechanism by which camptothecins inhibit the catalytic activity of Top1 (6 -8). Nevertheless, formation of CPT-Top1-DNA ternary complexes is insufficient to explain the cytotoxic effects of CPT (9 -11). Current models invoke interactions between ternary complexes and replication or transcriptional machinery as being important in conversion of ternary complexes to lethal forms of DNA damage (12, 13). However, it is not known whether physical interactions between Top1 and other proteins (including proteins involved in replication or transcription) are important in the cytotoxic effects of CPT. Recently, an interaction between Top1 and the SV40 T antigen helicase was shown to modulate formation of Top1-CPT-DNA ternary complexes, suggesting that similar interactions between Top1 and cellular helicases may mediate the cytotoxicity of CPT (14). In addition, Top1 rapidly redistributes from the nucleolus to the nucleus or cytoplasm and is degraded after cellular exposure to . These alterations may confer transient cellular resistance to CPT and could be mediated by interactions between Top1 and other proteins.Top1 is known to physically interact with the following proteins: HMG17 (20), casein kinase II (21), RNA polymerase I (22), nucleolin (23), SV40 T antigen (24,25), p53 (26,27), the TATA-binding protein (28), the splicing factors SF2/ASF (2...

We have attempted to identify human topoisomerase I-binding proteins in order to gain information regarding the cellular roles of this protein and the cytotoxic mechanisms of the anticancer drug camptothecin, which specifically targets topoisomerase I. In the course of this work we identified an interaction between the N-terminus of human topoisomerase I and the SV40 T antigen that is detectable in vitro using both affinity chromatography and co-immunoprecipitation. Additional results indicate that this interaction does not require intermediary DNA or stoichiometric quantities of other proteins. Furthermore, the interaction is detectable in vivo using a yeast two-hybrid assay. Two binding sites for T antigen are apparent on the topoisomerase I protein: one consisting of amino acids 1-139, the other present in the 383-765 region of the protein. Interestingly, nucleolin, which binds the 166-210 region of topoisomerase I, is able to bind an N-terminal fragment of topoisomerase I concurrently with T antigen. Taken together with our prior identification of nucleolin as a topoisomerase I-binding protein, the current results suggest that helicase-binding is a major role of the N-terminus of human topoisomerase I and that the resultant helicase-topoisomerase complex may function as a eukaryotic gyrase.

Camptothecins are broad-spectrum anticancer drugs that specifically target DNA topoisomerase I. Although the availability of camptothecins has had a significant impact on cancer therapeutics, de novo or acquired clinical resistance to camptothecins is common. Studies of camptothecin resistance using yeast and mammalian cell culture models suggest three general mechanisms of resistance: (1) reduced cellular accumulation of camptothecins, (2) alteration in the structure or location of topoisomerase I, and (3) alterations in the cellular response to camptothecin-DNA-ternary complex formation. The relevance of these mechanisms to clinical drug resistance is not yet known, but evaluation of these models in clinical specimens should enhance the use of camptothecins both as single agents and in combination with other anticancer drugs.

Confluent skin fibroblast cultures were prepared from 40 patients diagnosed with and surgically treated for an abdominal aortic aneurysm. An analysis of secreted type I and type III collagen in the media of these fibroblast preparations revealed reduced secretion of type III collagen from six patients. DNA sequence analysis of the entire coding domain of the pro alpha 1 (III) collagen mRNA in skin fibroblast RNA from these six patients revealed a C to T substitution at nucleotide 607 in one of the probands that would result in the replacement of a leucine residue with phenylalanine in the second position of the first tripeptide repeat in the triple-helical domain of type III collagen. Allele-specific hybridization analysis of genomic DNA from this proband and family members indicated that this non-glycine substitution probably contributed to the aneurysmal phenotype in this patient. No coding sequence mutations were found in the other five patients. It is clear from this study, therefore, that aberrant synthesis of type III collagen, as a consequence of both a coding sequence mutation and other factors contributing to reduced secretion of type III procollagen, will result in the development of an aortic aneurysm in a significant percentage of patients with this disease.