Assessment of DNA-PKcs kinase activity by quantum dot\u2013based microarray

Florian Lafont,Fabrice Fleury,Pierre Weigel,Nizar Ayadi,Cathy Charlier,Igor Nabiev,Houda Benhelli-Mokrani

doi:10.1038/s41598-018-29256-2

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.

Highlights

Understanding cellular systems requires identification and analysis of the functions of cellular components, especially their functional interrelation and regulation
Generation of the antibody microarray dedicated to the Non Homologous End Joining (NHEJ) DNA repair pathway. 16 antibodies and 4 control proteins were deposited in triplicate on the nitrocellulose membrane pad (Fig. 1)
We have found that the changes in the CPT-mediated phosphorylation level of RPA2 are less pronounced in the case of wortmannin compared to NU7441, which seems to act in a dose-dependent manner against DNA-PKcs activity (Fig. 5)

Summary

Introduction

Understanding cellular systems requires identification and analysis of the functions of cellular components, especially their functional interrelation and regulation. In response to DNA damage, active cell signaling pathways initiate a series of protein phosphorylation cascades. These processes involve a network of complex interactions of cellular components that coordinate the DNA Damage Response (DDR)[1]. Among DNA lesions, DSBs are the most toxic They can be repaired by two major mechanisms, Homologous Recombination (HR) and Non Homologous End Joining (NHEJ), wherein Rad[51] and DNA-PKcs, respectively, play a pivotal role[1]. C-region of DNA-PKcs contains a kinase domain, which is involved in its auto-phosphorylation and the phosphorylation of other proteins after DNA damage[2,5]. The spectral positions of the emission bands of different QDs widely vary, the peak wavelength depending on the QD composition and diameter (400 nm to 2 μm), whereas the fluorescence emission spectral width of each QD type is narrow[17,19]

Methods

Results

Conclusion