Assessment of DNA-PKcs kinase activity by quantum dot–based microarray

Abstract

Therapeutic efficacy against cancer is often based on a variety of DNA lesions, including DNA double-strand breaks (DSBs) which are repaired by homologous recombination and non-homologous end joining (NHEJ) pathways. In the past decade, the functions of the DNA repair proteins have been described as a potential mechanism of resistance in tumor cells. Therefore, the DNA repair proteins have become targets to improve the efficacy of anticancer therapy. Given the central role of DNA-PKcs in NHEJ, the therapeutic efficacy of targeting DNA-PKcs is frequently described as a strategy to prevent repair of treatment-induced DNA damage in cancer cells. The screening of a new inhibitor acting as a sensitizer requires the development of a high-throughput tool in order to identify and assess the most effective molecule. Here, we describe the elaboration of an antibody microarray dedicated to the NHEJ pathway that we used to evaluate the DNA-PKcs kinase activity in response to DNA damage. By combining a protein microarray with Quantum-Dot detection, we show that it is possible to follow the modification of phosphoproteomic cellular profiles induced by inhibitors during the response to DNA damage. Finally, we discuss the promising tool for screening kinase inhibitors and targeting DSB repair to improve cancer treatment.

Spatial Bias in Antibody Microarrays May Be an Underappreciated Source of Variability

“…Spatial bias in antibody microarrays remains understudied and underappreciated. Replicate spot randomization and spatial variation have only been mentioned, but not characterized, in dual-color antibody arrays. , Recently published antibody microarray protocols ,,, neither mention spatial bias nor randomization of replicate spots. To the best of our knowledge, there has been no quantitative characterization of spatial bias in antibody microarrays using replicate spots and, specifically, of the effect of spatial bias on assay performance and reproducibility.…”