A simple microscopy setup for visualizing cellular responses to DNA damage at particle accelerator facilities

Sytze Brandenburg,Emiel R Van Der Graaf,Christoph Meyer,Ron A Hoebe,Haibin Qian,Marc Jan Van Goethem,Harry Kiewiet,Przemek M Krawczyk,Michel R Faas

doi:10.1038/s41598-021-92950-1

Abstract

Cellular responses to DNA double-strand breaks (DSBs) not only promote genomic integrity in healthy tissues, but also largely determine the efficacy of many DNA-damaging cancer treatments, including X-ray and particle therapies. A growing body of evidence suggests that activation of the mechanisms that detect, signal and repair DSBs may depend on the complexity of the initiating DNA lesions. Studies focusing on this, as well as on many other radiobiological questions, require reliable methods to induce DSBs of varying complexity, and to visualize the ensuing cellular responses. Accelerated particles of different energies and masses are exceptionally well suited for this task, due to the nature of their physical interactions with the intracellular environment, but visualizing cellular responses to particle-induced damage - especially in their early stages - at particle accelerator facilities, remains challenging. Here we describe a straightforward approach for real-time imaging of early response to particle-induced DNA damage. We rely on a transportable setup with an inverted fluorescence confocal microscope, tilted at a small angle relative to the particle beam, such that cells can be irradiated and imaged without any microscope or beamline modifications. Using this setup, we image and analyze the accumulation of fluorescently-tagged MDC1, RNF168 and 53BP1—key factors involved in DSB signalling—at DNA lesions induced by 254 MeV α-particles. Our results provide a demonstration of technical feasibility and reveal asynchronous initiation of accumulation of these proteins at different individual DSBs.

Highlights

Cellular responses to DNA double-strand breaks (DSBs) promote genomic integrity in healthy tissues, and largely determine the efficacy of many DNA-damaging cancer treatments, including X-ray and particle therapies
Emerging evidence indicates that cellular responses to DSBs are at least partly determined by their complexity, i.e. the number, structure and distribution of clustered DNA lesions, such as strand cross-links, base/nucleotide alterations or nicks[4]
These complex DSBs pose a considerable challenge to the repair machinery, require extensive processing by various enzymes, and have enhanced relative biological effectiveness (RBE) in inducing genomic rearrangements or cell death, as compared

Summary

Introduction

Cellular responses to DNA double-strand breaks (DSBs) promote genomic integrity in healthy tissues, and largely determine the efficacy of many DNA-damaging cancer treatments, including X-ray and particle therapies. DNA double-strand breaks (DSBs) are arguably the most severe, frequently leading to cell death, potentially oncogenic mutations or chromosome rearrangements, if not repaired timely and correctly[3] These dangerous consequences make DSBs a potent, but double-edged sword: on the one hand, DSB-inducing agents power many among the most effective cancer therapies; on the other hand, DSBs can initiate or contribute to the deterioration of genetic material and to carcinogenesis. On the other end of the complexity spectrum are the DSBs generated by accelerated, high-LET, heavy particles, which are often accompanied by multiple other lesions, including DSBs, often in close (nanometer-scale) p roximity[5] These complex DSBs pose a considerable challenge to the repair machinery, require extensive processing by various enzymes, and have enhanced relative biological effectiveness (RBE) in inducing genomic rearrangements or cell death, as compared. In spite of these important developments, our understanding of cellular responses to DNA lesions of varying complexity is still limited and methods for studying this aspect of DNA damage response in detail are urgently needed

Methods

Results

Conclusion