Biological Imaging of Heavy Charged-Particle Tracks

Abstract

The immunocytochemical response to DNA damage induced by low-energy bismuth and carbon ions was investigated in normal human fibroblasts. Inside the nuclei, the traversing charged particles lead to the accumulation of proteins related to DNA lesions and repair along the ion trajectories. Irradiation under a standard geometric setup with the beam direction perpendicular to the cell monolayer generates spots of these proteins as described previously for MRE11B (hMre11), CDKN1A (p21) and PCNA (Jakob et al., Int. J. Radiat. Biol. 78, 75-88, 2002). Here we present data obtained with a new irradiation geometry characterized by a small angle between the beam direction and the monolayer of cells. This new irradiation geometry leads to the formation of protein aggregates in the shape of streaks stretching over several micrometers in the x/y plane, thus facilitating the analysis of the fluorescence distributions along the particle trajectories. Measurements of fluorescence intensity along the ion tracks in double- and triple-stained samples revealed a strict spatial correlation for the occurrence of CDKN1A and MRE11B clusters. In addition, immunostained gamma-H2AX is used as a marker of double-strand breaks (DSBs) to visualize the localized induction of these lesions along the particle paths. A clear coincidence of CDKN1A and gamma-H2AX signals within the ion-induced streaks is observed. Also for PCNA, which mainly associates with lesions processed by excision repair, a strict colocalization with the MRE11B aggregations was found along the ion trajectories, despite the higher estimated yield of this type of lesions compared to DSBs. Strikingly similar patterns of protein clusters are generated not only for the various proteins studied but also using different ion species from carbon to bismuth, covering LET values ranging from about 300 to 13600 keV/microm and producing estimated DSB densities differing by a factor around 45. The patterns of protein clustering along the very heavy-ion trajectories appear far more heterogeneous than expected based on idealized DSB distributions arising from model calculations. The results suggest that additional factors like compaction or confined movement of chromatin are responsible for the observed clustering of proteins.

Activation of ATM and Chk2 kinases in relation to the amount of DNA strand breaks

“…As the average number of g-H2AX foci per cell well compares with the number of induced DSBs (Rogakou et al, 1999;Petersen et al, 2001;Furuta et al, 2003;Jakob et al, 2003;Rothkamm and Lobrich, 2003), we quantitated DSBs in exponentially growing cells by enumerating g-H2AX foci. In LCL-N, the basal number of foci per cells accounted for 2.870.9, but 5 min after irradiation with 0.25, 0.5 and 1 Gy their number rose to 7.870.4,13.670.8 and 19.971,respectively (Figure 1b).…”

PCNA-dependent accumulation of CDKN1A into nuclear foci after ionizing irradiation

“…For CDKN1A detection, cells were permeabilized in Hepes buffer before fixation as described [33]. PCNA immunostaining was performed as described in [34] using cytoskeleton buffer for pre-extraction. The following primary antibodies were used: mouse anti CIP1/WAF1 (BD; dilution 1:80); mouse anti PCNA (Chemicon; 1:50); mouse/rabbit anti γH2AXser139 (Millipore; 1:500); mouse/rabbit anti 53BP1 (Bethyl Laboratories; 1:2000) and rabbit anti XRCC1 (Alexis Biochemicals; 1:1000).…”

“…Microscopic analysis of fixed samples was carried out on a confocal microscope as described previously [31,34]. A Leica TCS confocal laser scanning system equipped with a DM IRBE inverted microscope (lens: Plan Apo 63× / 1.32 oil) and an argon/krypton laser were used.…”

“…In addition, we previously reported on the colocalization of CDKN1A and PCNA foci at the sites of charged particle traversal [34]. To test whether CDKN1A and PCNA foci would colocalize in response to low LET ionizing radiation (IR), we exposed G0/G1 AG1522C fibroblasts to 8 Gy X-rays and fixed and stained nuclei for both CDKN1A and PCNA, as previously described (BJ, RR, 2003).…”

“…particle traversal [34]. However, it must be considered that DNA damage induced by high linear energy (LET) charged particle irradiation, besides predominantly DNA double-strand breaks (DSBs), likely additionally contains in close proximity multiple other lesions, including damaged DNA bases and DNA single-strand breaks (SSBs) [35].…”