Assessment of DNA Double-Strand Break Repair at Laser-Induced Damage Sites in Hematological Cancer Cells

Abstract

Certain tumors rely heavily on their DNA repair capability to survive the DNA damage induced by chemotherapeutic agents. Therefore, it is important to monitor the dynamics of DNA repair in patient samples during the course of their treatment, in order to determine whether a particular drug regimen perturbs the DNA repair networks in cancer cells and provides therapeutic benefits. Quantitative measurement of proteins and/or their post-translational modification(s) at DNA double strand breaks (DSBs) induced by laser micro-irradiation provides an applicable diagnostic approach to examine DNA repair and its dynamics. However, its use is restricted to adherent cell lines and not employed in suspension tumor cells that include the many hematological malignancies. Here, we now report a method to laser micro-irradiate and quantitatively measure DNA repair transactions at DSB sites in a variety of suspension leukemia and lymphoma cells including primary patient samples. We have also used our refined micro-laser method to examine the effects of small molecule inhibitors of epigenetic histone-modifying enzymes in modulating the levels of histone H3 lysine-27 trimethylation (H3K27me3) at DSB sites during DNA repair in a subset of DNA repair 'addicted' diffuse large B-cell lymphoma (DLBCL) cells. Results from our mechanistic studies and proof-of-principle data with patient samples together show the effectiveness of using the modified micro-laser-based assay to examine DNA repair directly in suspension cancer cells, and has important clinical implications by serving as a valuable tool to assess drug efficacies in cancer cells that grow in suspension. Funding: This work was supported by National Institute of Health grants (1R011CA188520 and R01CA1885200S1) as well as Huntsman Cancer Institute and Radiation Oncology development funds to SB. Declaration of Interest: The authors have no competing financial interests. Ethical Approval: The patient sample culture was carried out in accordance with the University of Utah IRB guidelines. The samples are de-identified samples and hence we do not have access to the patient history.