Abstract
Quantification of the DNA damage induced by chemotherapy in patient cells may aid in personalization of the dose used. However, assays to evaluate individual patient response to chemotherapy are not available today. Here, we present an assay that quantifies single-stranded lesions caused by the chemotherapeutic drug Bleomycin (BLM) in peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals. We use base excision repair (BER) enzymes to process the DNA damage induced by BLM and then extend the processed sites with fluorescent nucleotides using a DNA polymerase. The fluorescent patches are quantified on single DNA molecules using fluorescence microscopy. Using the assay, we observe a significant variation in the in vitro induced BLM damage and its repair for different individuals. Treatment of the cells with the BER inhibitor CRT0044876 leads to a lower level of repair of BLM-induced damage, indicating the ability of the assay to detect a compromised DNA repair in patients. Overall, the data suggest that our assay could be used to sensitively detect the variation in BLM-induced DNA damage and repair in patients and can potentially be able to aid in personalizing patient doses.
Highlights
Chemotherapy drugs kill proliferating cancer cells by targeting DNA, RNA [1], enzymes [2] and proteins [3]
We have shown that the same assay can be extended to ionizing radia tion and hyperthermia induced single strand DNA damage on peripheral blood mononuclear cells (PBMCs) derived from healthy individuals [37]
We demonstrate base excision repair (BER) inhibition caused by the APE1 inhibitor CRT0044876, which we propose as a model for identifying individuals with defective DNA repair pathways
Summary
Chemotherapy drugs kill proliferating cancer cells by targeting DNA, RNA [1], enzymes [2] and proteins [3]. The drugs can be classified based on their ability to induce different forms of DNA damage, the most common being single-strand breaks (SSBs), double-strand breaks (DSBs) and DNA-protein crosslinks [4]. Differences in individual normal tissue sensitivity results in variation in the severity of side effects and is a challenge to optimal dosing of chemotherapy. Previous exposure to radiation or chemotherapy, gender, rate of metabolism of chemotherapy drugs and other genetic traits can affect the outcome of chemotherapy [8,9]. Despite the broad use of chemotherapy, there are no clinically available assays that can predict patient’s hypersensitivity or resistance towards specific chemotherapy drugs
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