Nanostructure of Clustered DNA Damage in Leukocytes after In-Solution Irradiation with the Alpha Emitter Ra-223.

Harry Scherthan,Felix Bestvater,Matthias Port,Sarah Schumann,Emanuel Maus,Michael Lassmann,Razan Muhtadi,Robert Chojowski,Jin-Ho Lee,Michael Hausmann

doi:10.3390/cancers11121877

Abstract

Background: Cancer patients are increasingly treated with alpha-particle-emitting radiopharmaceuticals. At the subcellular level, alpha particles induce densely spaced ionizations and molecular damage. Induction of DNA lesions, especially clustered DNA double-strand breaks (DSBs), threatens a cell’s survival. Currently, it is under debate to what extent the spatial topology of the damaged chromatin regions and the repair protein arrangements are contributing. Methods: Super-resolution light microscopy (SMLM) in combination with cluster analysis of single molecule signal-point density regions of DSB repair markers was applied to investigate the nano-structure of DNA damage foci tracks of Ra-223 in-solution irradiated leukocytes. Results: Alpha-damaged chromatin tracks were efficiently outlined by γ-H2AX that formed large (super) foci composed of numerous 60–80 nm-sized nano-foci. Alpha damage tracks contained 60–70% of all γ-H2AX point signals in a nucleus, while less than 30% of 53BP1, MRE11 or p-ATM signals were located inside γ-H2AX damage tracks. MRE11 and p-ATM protein fluorescent tags formed focal nano-clusters of about 20 nm peak size. There were, on average, 12 (±9) MRE11 nanoclusters in a typical γ-H2AX-marked alpha track, suggesting a minimal number of MRE11-processed DSBs per track. Our SMLM data suggest regularly arranged nano-structures during DNA repair in the damaged chromatin domain.

Highlights

Ionizing radiation (IR) transfers energy to electrons of atoms and molecules, leading to their ionization
(Figure 1A). γ-H2AX formation is largely dependent on the DNA double strand break (DSB)-responsive kinase ATM that in its active form is phosphorylated at Serin-1981 [48], which depends on complex formation with NBS1 [49]
We found that 53BP1 and MRE11 signals were located in the non-irradiated nuclear environment, while their frequency was enriched in the alpha-damaged chromatin tracks, suggesting that the damage caused by a single alpha particle did not deplete the protein pool of a hit nucleus, whereas 53BP1 depletion was observed after repeated C

Summary

Introduction

Ionizing radiation (IR) transfers energy to electrons of atoms and molecules, leading to their ionization. E.g., alpha-emitting radionuclides displays high linear energy transfer (LET) to the molecular and supra-molecular environment along a particle track through the cell. Alpha particles considered for treatment have a range of up to 90 μm in water [6] and they have a kinetic energy of about 1.4 to 2.2 MeV per nucleon. They pass cells with a mean LET of about. Methods: Super-resolution light microscopy (SMLM) in combination with cluster analysis of single molecule signal-point density regions of DSB repair markers was applied to investigate the nano-structure of DNA damage foci tracks of Ra-223 in-solution irradiated leukocytes. There were, on average, 12 (±9) MRE11 nanoclusters in a typical γ-H2AX-marked alpha track, suggesting a minimal number of MRE11-processed DSBs per track

Methods

Results

Discussion

Conclusion