Novel actions of inhibitors of dna topoisomerase i

William T Beck, PhD12,Yin-Yuan Mo, PhD1, P L Rachel 2, BS Rajiv Patel1

1Departments of Molecular Genetics1 and Pharmaceutics and Pharmacodynamics 2, The University of Illinois at Chicago, Chicago, IL USA

AIMS Our overall aim is to better understand the molecular actions by which topoisomerase inhibitors cause tumor cell cytotoxicity. The DNA topoisomerases are nuclear enzymes that catalyze the breakage and rejoining of DNA to relieve torsional stress during replication and transcription. The type I enzymes cause single strand breaks following covalent binding to the 3 terminus and relieve torsional stress on DNA in an ATP-independent manner. While non-essential in yeast, topo I is essential and required for mammalian cell survival, and is involved in chromatin remodeling, DNA relaxation, transcription, chromosome condensation, and possibly DNA repair. Inhibitors of topo I such as topotecan (TPT) and irinotecan are clinically important in cancer chemotherapy, and stabilize DNA-topo I complexes by blocking DNA religation. METHODS and RESULTS We have examined the actions of topo I inhibitors on the activity, subcellular distribution and protein modification of topo I. We and others have shown that topo I is sumoylated in response to TPT; moreover, we found that treatment with TPT causes topo I to leave the nucleolus (nucleolar delocalization; NDL). NDL by topo I inhibitors is attenuated in tumor cell lines resistant to these agents, suggesting a relationship between topo I NDL and drug responsiveness. Experiments with enhanced green fluorescent protein (EGFP)-topo I fusion proteins revealed a relationship between sumoylation of topo I and its NDL. Because UBC9 is an essential conjugating enzyme for sumoylation, we examined the role of UBC9 in the sumoylation and NDL of topo I. Overexpression of a dominant-negative UBC9 (DN-UBC9) not only reduced sumoylation of topo I, but also attenuated its NDL, suggesting that UBC9/SUMO plays a role in the NDL of topo I. To define the UBC9 binding sites in topo I, we introduced various deletions of topo I-EGFP constructs into HeLa cells. By GST-UBC9 pull-down experiments and immunoprecipitation, we identified two potential UBC9 binding sites on topo I, both of which reside at the N-termimus. The major UBC9 binding site was identified to be within aa 148-187 in topo I. By GST-topo I pull-down experiments, we also found that cysteine 93 of UBC9, an active site for sumoylation, is not required for its binding to topo I. The potential relevance of UBC9 may relate to the following: we have found that in ovarian carcinoma cell lines, UBC9 levels appear to be related to the sensitivity of the cells to cis-platin. Moreover, we transfected HeLa and MCF-7 cells with the DN-UBC9 construct and found that it made the cells more sensitive to DNA damaging agents such as TPT, suggesting a role for UBC9 in drug responsiveness. CONCLUSIONS Overall, our results suggest that inhibitors of topoisomerases initiate cytotoxic signaling that involves enzyme redistribution and protein modification. Sumoylation of topo I is associated with its nucleolar delocalization and occurs in response to treatment with inhibitors of topo I; this requires UBC9. That DN-UBC9 attenuates sumoylation of topo I and increases the sensitivity of cells to topo I inhibitors, suggests an effect on DNA metabolism and possibly repair processes, and experiments are in progress to better understand this relationship. (Supported by grants from the National Cancer Institute to WTB)

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Novel Molecular Therapies.