Beyond Repair Foci: DNA Double-Strand Break Repair in Euchromatic and Heterochromatic Compartments Analyzed by Transmission Electron Microscopy

DNA double-strand breaks (DSBs) generated by ionizing radiation pose a serious threat to the preservation of genetic and epigenetic information. The known importance of local chromatin configuration in DSB repair raises the question of whether breaks in different chromatin environments are recognized and repaired by the same repair machinery and with similar efficiency. An essential step in DSB processing by non-homologous end joining is the high-affinity binding of Ku70-Ku80 and DNA-PKcs to double-stranded DNA ends that holds the ends in physical proximity for subsequent repair.Using transmission electron microscopy to localize gold-labeled pKu70 and pDNA-PKcs within nuclear ultrastructure, we monitored the formation and repair of actual DSBs within euchromatin (electron-lucent) and heterochromatin (electron-dense) in cortical neurons of irradiated mouse brain.While DNA lesions in euchromatin (characterized by two pKu70-gold beads, reflecting the Ku70-Ku80 heterodimer) are promptly sensed and rejoined, DNA packaging in heterochromatin appears to retard DSB processing, due to the time needed to unravel higher-order chromatin structures. Complex pKu70-clusters formed in heterochromatin (consisting of 4 or ≥ 6 gold beads) may represent multiple breaks in close proximity caused by ionizing radiation of highly-compacted DNA. All pKu70-clusters disappeared within 72 hours post-irradiation, indicating efficient DSB rejoining. However, persistent 53BP1 clusters in heterochromatin (comprising ≥ 10 gold beads), occasionally co-localizing with γH2AX, but not pKu70 or pDNA-PKcs, may reflect incomplete or incorrect restoration of chromatin structure rather than persistently unrepaired DNA damage.Higher-order organization of chromatin determines the accessibility of DNA lesions to repair complexes, defining how readily DSBs are detected and processed. DNA lesions in heterochromatin appear to be more complex, with multiple breaks in spatial vicinity inducing severe chromatin disruptions. Imperfect restoration of chromatin configurations may leave DSB-induced epigenetic memory of damage with potentially pathological repercussions

Targeted Large-Volume Lymphocyte Removal Using Magnetic Nanoparticles in Blood Samples of Patients with Chronic Lymphocytic Leukemia: A Proof-of-Concept Study

In the past, our research group was able to successfully remove circulating tumor cells with magnetic nanoparticles. While these cancer cells are typically present in low numbers, we hypothesized that magnetic nanoparticles, besides catching single cells, are also capable of eliminating a large number of tumor cells from the blood ex vivo. This approach was tested in a small pilot study in blood samples of patients suffering from chronic lymphocytic leukemia (CLL), a mature B-cell neoplasm. Cluster of differentiation (CD) 52 is a ubiquitously expressed surface antigen on mature lymphocytes. Alemtuzumab (MabCampath®) is a humanized, IgG1κ, monoclonal antibody directed against CD52, which was formerly clinically approved for treating chronic lymphocytic leukemia (CLL) and therefore regarded as an ideal candidate for further tests to develop new treatment options. Alemtuzumab was bound onto carbon-coated cobalt nanoparticles. The particles were added to blood samples of CLL patients and finally removed, ideally with bound B lymphocytes, using a magnetic column. Flow cytometry quantified lymphocyte counts before, after the first, and after the second flow across the column. A mixed effects analysis was performed to evaluate removal efficiency. p n = 10), using a fixed nanoparticle concentration, CD19-positive B lymphocytes were reduced by 38% and by 53% after the first and the second purification steps (p = 0.002 and p = 0.005), respectively. In a second patient cohort (n = 11), the nanoparticle concentration was increased, and CD19-positive B lymphocytes were reduced by 44% (p 20 G/L), an improved efficiency of approximately 20% was observed using higher nanoparticle concentrations. A 40 to 50% reduction of B lymphocyte count using alemtuzumab-coupled carbon-coated cobalt nanoparticles is feasible, also in patients with a high lymphocyte count. A second purification step did not further increase removal. This proof-of-concept study demonstrates that such particles allow for the targeted extraction of larger amounts of cellular blood components and might offer new treatment options in the far future

Quantification of gold-labeled pKu70 and 53BP1 in cortical neurons analyzed by TEM. Induction.

<p>Quantification of pKu70 and 53BP1 cluster in euchromatic and heterochromatic domains at 5<b> </b>min and 40<b> </b>min after irradiation with doses ranging from 1 to 10 Gy. The induction of pKu70 (<b>A</b>) and 53BP1 cluster (<b>B</b>) was clearly dependent on the radiation dose, with a linear correlation in the dose range of 1 to 10 Gy. <b>Repair.</b> Quantification of pKu70 (<b>C</b>) and 53BP1 cluster (<b>D</b>) per nucleus at 5<b> </b>min, 20<b> </b>min, 40<b> </b>min, 5 h, 24 h, 48 h, and 72 h after irradiation (6Gy). 53BP1 clusters disappeared with kinetics similar to those observed with heterochromatin-associated pKu70 clusters.</p

Gold-labeled pKu70 and 53BP1 clusters in cortical neurons analyzed at different time-points after irradiation.

<p>TEM micrographs of double-labeling of pKu70 (10-nm beads) and pDNA-PKcs or 53BP1 (6-nm beads) at different magnifications. (<b>A</b>) All pKu70 clusters consisting of 2 (upper panel) or more gold beads (lower panel) co-localize with pDNA-PKcs, suggesting that these lesions are actively processed DSBs (analyzed at 20<b> </b>min and 24 h post-irradiation). (<b>B</b>) In contrast, huge 53BP1 clusters in heterochromatin at late repair-times (72 h post-irradiation) do not co-localize with pKu70, suggesting that these lesions do not reflect persistently unrepaired DSBs. (pKu70 beads are marked by red arrows).</p

Quantification of the different pKu70 and 53BP1 clusters in euchromatic and heterochromatic compartments of cortical neurons analyzed by TEM.

<p>(<b>A</b>) Quantification of pKu70 clusters consisting of 1, 2, 4, or ≥6 beads, analyzed separately in euchromatic and heterochromatic compartments at the different time-points after irradiation (6 Gy). We observed solely pKu70 clusters of 1 and 2 beads in euchromatin, but increasingly complex pKu70 clusters with 4 or ≥6 beads in heterochromatin. (<b>B</b>) Quantification of 53BP1 clusters <10 and ≥10 beads, analyzed in the heterochromatic compartment at different time-points after irradiation (6Gy). We observed huge 53BP1 clusters at late repair-times.</p

Gold-labeled pKu70 and 53BP1 in cortical neurons of brain analyzed 40 min after irradiation with 6Gy.

<p>TEM micrographs of double-labeling of pKu70 (10-nm beads) and p53BP1 (6-nm beads) at different magnifications (boxed regions are shown at higher magnifications in the following images). Complex pKu70 clusters (consisting of 4 gold beads) co-localizing with 53BP1 were observed in heterochromatic regions, but only isolated pKu70 clusters without any p53BP1 binding were observed in euchromatic regions (pKu70 beads are marked by red arrows).</p

Gold-labeled pKu70 and 53BP1 in mouse tissues and human fibroblasts analyzed 40 min after irradiation with 6Gy.

<p>TEM micrographs of double-labeling of pKu70 (10-nm beads) and p53BP1 (6-nm beads) at different magnifications. In enterocytes of small intestine, keratinocytes of skin as well as in human fibroblast, radiation-induced pKu70 clusters (consisting of 2 gold beads, red arrows) co-localize with 53BP1 only in heterochromatic regions.</p