Biological imaging of heavy charged-particle tracks.
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.
Jakob
B
B
Gesellschaft für Schwerionenforschung, Biophysik, Planckstrasse 1, D-64291 Darmstadt, Germany. B.Jakob@gsi.de
Scholz
M
M
Taucher-Scholz
G
G
D001729:Bismuth; D002244:Carbon; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000032:analysis; D004247:DNA / Q000528:radiation effects*; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000032:analysis; D005347:Fibroblasts / Q000528:radiation effects; D020450:Heavy Ions; D006657:Histones / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D018499:Linear Energy Transfer; D000076228:MRE11 Homologue Protein; D010766:Phosphorylation; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; C121766:MRE11 protein, human; D018809:Proliferating Cell Nuclear Antigen; D002244:Carbon; D004247:DNA; D000076228:MRE11 Homologue Protein; D001729:Bismuth