Abstract
DNA-damaging agents, such as radiation and chemotherapy, are common in cancer treatment, but the dosing has proven to be challenging, leading to severe side effects in some patients. Hence, to be able to personalize DNA-damaging chemotherapy, it is important to develop fast and reliable methods to measure the resulting DNA damage in patient cells. Here, we demonstrate how single DNA molecule imaging using fluorescence microscopy can quantify DNA-damage caused by the topoisomerase II (TopoII) poison etoposide. The assay uses an enzyme cocktail consisting of base excision repair (BER) enzymes to repair the DNA damage caused by etoposide and label the sites using a DNA polymerase and fluorescently labeled nucleotides. Using this DNA-damage detection assay we find a large variation in etoposide induced DNA-damage after in vitro treatment of blood cells from healthy individuals. We furthermore used the TopoII inhibitor ICRF-193 to show that the etoposide-induced damage in DNA was TopoII dependent. We discuss how our results support a potential future use of the assay for personalized dosing of chemotherapy.
Highlights
The treatment of cancer includes DNA-damaging agents like radiation and chemotherapy [1]
Detection of DNA damage caused by etoposide
The protocol for quantifying single-strand lesions caused by etoposide and fluorescent labeling of the damaged sites is schematically outlined in Fig. 1 A [29,30]
Summary
The treatment of cancer includes DNA-damaging agents like radiation and chemotherapy [1]. Chemotherapeutic drugs, such as cisplatin, doxorubicin and etoposide, induce DNA damage of various forms, such as double-strand breaks (DSBs), single-strand breaks (SSBs), crosslinks and damaged bases [2]. Methods to measure and monitor interindividual variation in chemotherapy response are still lacking, which has resulted in a crude dosing model [13]. This in turn means that while some patients are underdosed so that there is little effect of the drug, other patients experience severe side effects. There is a need for methods to personalize cancer therapy
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