Elucidation of the Clustered Nano-Architecture of Radiation-Induced DNA Damage Sites and Surrounding Chromatin in Cancer Cells: A Single Molecule Localization Microscopy Approach

Abstract

In cancer therapy, the application of (fractionated) harsh radiation treatment is state of the art for many types of tumors. However, ionizing radiation is a “double-edged sword”—it can kill the tumor but can also promote the selection of radioresistant tumor cell clones or even initiate carcinogenesis in the normal irradiated tissue. Individualized radiotherapy would reduce these risks and boost the treatment, but its development requires a deep understanding of DNA damage and repair processes and the corresponding control mechanisms. DNA double strand breaks (DSBs) and their repair play a critical role in the cellular response to radiation. In previous years, it has become apparent that, beyond genetic and epigenetic determinants, the structural aspects of damaged chromatin (i.e., not only of DSBs themselves but also of the whole damage-surrounding chromatin domains) form another layer of complex DSB regulation. In the present article, we summarize the application of super-resolution single molecule localization microscopy (SMLM) for investigations of these structural aspects with emphasis on the relationship between the nano-architecture of radiation-induced repair foci (IRIFs), represented here by γH2AX foci, and their chromatin environment. Using irradiated HeLa cell cultures as an example, we show repair-dependent rearrangements of damaged chromatin and analyze the architecture of γH2AX repair clusters according to topological similarities. Although HeLa cells are known to have highly aberrant genomes, the topological similarity of γH2AX was high, indicating a functional, presumptively genome type-independent relevance of structural aspects in DSB repair. Remarkably, nano-scaled chromatin rearrangements during repair depended both on the chromatin domain type and the treatment. Based on these results, we demonstrate how the nano-architecture and topology of IRIFs and chromatin can be determined, point to the methodological relevance of SMLM, and discuss the consequences of the observed phenomena for the DSB repair network regulation or, for instance, radiation treatment outcomes.

Focused Ion Microbeam Irradiation Induces Clustering of DNA Double-Strand Breaks in Heterochromatin Visualized by Nanoscale-Resolution Electron Microscopy

“…Previous reports using super-resolution fluorescence microscopy have shown the formation of clustered DNA repair foci along the particle tracks in heavy ion-irradiated cells [ 21 , 22 ]. Super-resolution fluorescence microscopy has managed to overcome the diffraction limit, thereby enabling the imaging of cellular structures with an improved resolution for many applications.…”

On the Equivalence of the Biological Effect Induced by Irradiation of Clusters of Heavy Atom Nanoparticles and Homogeneous Heavy Atom-Water Mixtures

“…Furthermore, one could take a linear combination of I 1 nuc and I 1 cyt corresponding to the different weighting factors for the nuclear and cytoplasmic targets and still have nearly equivalent results for the MixNP and full cluster cases. Hence, the conclusion regarding the equivalence of the biological effect in these two scenarios is robust, even though there is ongoing controversy regarding the nature of the critical targets, as was highlighted in the introduction [ 12 , 13 , 14 , 15 ].…”

“…The effects of nanoparticles on DNA are currently a matter for discussion in the scientific literature, see e.g., [ 12 , 13 ]. Furthermore, it is not clear that the only critical subcellular target is nuclear DNA.…”

Heterochromatin Networks: Topology, Dynamics, and Function (a Working Hypothesis)

“…The mean diameter of the “alveoli” in SkBr3 cells was 1.7 µm. Deeper insight into folding can also be quantitatively obtained using the loci data set as visualized in Figure 2 c. The application of cluster algorithms [ 59 ] defines the core clusters within the heterochromatin areas ( Figure 2 d). The results shown in Figure 2 c,d indicate that the highly dense regions of CHR visualized by standard microscopy included lower-level subdomains.…”