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.
Highlights
DNA-modifying enzymes have been used for many years as drug targets in chemotherapeutic anticancer therapy, which exploits the high transcription and replication rates of cancer cells
Caco2 has eight copies of the hTOPI gene, whereas HT29 has five [27]. These cell lines were examined with regards to hTopI protein amount, hTopI activity, cellular CPT sensitivity, tyrosyl-DNA phosphodiesterase 1 (TDP1) activity, and CPT response of hTopI in whole cell extracts
When comparing Caco2 and HT29, we observed that the Caco2 cell line containing close to twofold the amount of hTOPI genes compared to HT29 had an approximate twofold increase in protein amount and hTopI activity
Summary
DNA-modifying enzymes have been used for many years as drug targets in chemotherapeutic anticancer therapy, which exploits the high transcription and replication rates of cancer cells. CPT poisons the cells mainly through the generation of double-stranded DNA breaks caused by S-phase specific collision of replication forks with the hTopI-DNA complexes [11]. HTopI has been widely evaluated as a predictive biomarker for CPT-based therapy both at gene-, mRNA-, protein-, and activity level with somewhat diverging results. We show here that direct determination of the drug response of hTopI is a better predictive marker for cellular CPT sensitivity than looking solely at gene copy number, mRNA amount, protein amount, or hTopI activity without drug. Since other factors than hTopI have been shown to influence CPT response we suggest that additional assays, e.g., measurement of TDP1 activity may be included
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