title: Topoisomerase I as a biomarker: detection of activity at the single molecule level.
abstract: Human topoisomerase I (hTopI) is an essential cellular enzyme. The enzyme is often upregulated in cancer cells, and it is a target for chemotherapeutic drugs of the camptothecin (CPT) family. Response to CPT-based treatment is dependent on hTopI activity, and reduction in activity, and mutations in hTopI have been reported to result in CPT resistance. Therefore, hTOPI gene copy number, mRNA level, protein amount, and enzyme activity have been studied to explain differences in cellular response to CPT. We show that Rolling Circle Enhanced Enzyme Activity Detection (REEAD), allowing measurement of hTopI cleavage-religation activity at the single molecule level, may be used to detect posttranslational enzymatic differences influencing CPT response. These differences cannot be detected by analysis of hTopI gene copy number, mRNA amount, or protein amount, and only become apparent upon measuring the activity of hTopI in the presence of CPT. Furthermore, we detected differences in the activity of the repair enzyme tyrosyl-DNA phosphodiesterase 1, which is involved in repair of hTopI-induced DNA damage. Since increased TDP1 activity can reduce cellular CPT sensitivity we suggest that a combined measurement of TDP1 activity and hTopI activity in presence of CPT will be the best determinant for CPT response.
authors.affiliation: Department of Pathology, Aarhus University Hospital, Aarhus C 8000, Denmark. joanna.proszek@gmail.com.
authors.forename: Joanna
authors.initials: J
authors.lastname: Proszek
authors.affiliation: Department of Pathology, Aarhus University Hospital, Aarhus C 8000, Denmark. royamit79@yahoo.com.
authors.forename: Amit
authors.initials: A
authors.lastname: Roy
authors.affiliation: Department of Pathology, Aarhus University Hospital, Aarhus C 8000, Denmark. annkatrinejakobsen@gmail.com.
authors.forename: Ann-Katrine
authors.initials: AK
authors.lastname: Jakobsen
authors.affiliation: Department of Pathology, Aarhus University Hospital, Aarhus C 8000, Denmark. rff@mb.au.dk.
authors.forename: Rikke
authors.initials: R
authors.lastname: Frøhlich
authors.affiliation: Department of Pathology, Aarhus University Hospital, Aarhus C 8000, Denmark. brk@mb.au.dk.
authors.forename: Birgitta R
authors.initials: BR
authors.lastname: Knudsen
authors.affiliation: Department of Pathology, Aarhus University Hospital, Aarhus C 8000, Denmark. magnstou@rm.dk.
authors.forename: Magnus
authors.initials: M
authors.lastname: Stougaard
mesh_terms: D015415:Biomarkers; D015153:Blotting, Western; D018938:Caco-2 Cells; D002166:Camptothecin / Q000494:pharmacology; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D018628:Gene Dosage / Q000235:genetics; D019073:HT29 Cells; D006801:Humans; D009154:Mutation; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism
chemical_list: D015415:Biomarkers; D010727:Phosphoric Diester Hydrolases; C498508:TDP1 protein, human; D004264:DNA Topoisomerases, Type I; C528909:TOP1 protein, human; D002166:Camptothecin