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. 2015 Jul;185(7):2049–2060. doi: 10.1016/j.ajpath.2015.03.014

Figure 4.

Figure 4

Association of chromosome rearrangement and DNA damage in fusion-derived cells. A: Representative image of chromosomal aberrations. The metaphase chromosome spread was prepared from fusion-derived clone (Fc) 15 at passage 11. The arrowhead points to fusion between the long arms of two acrocentric chromosomes (ie, Robertsonian translocation); arrows denote chromosome breaks. Robertsonian translocations were observed in 9 (11%) of 83 cells and chromosomal breaks and translocation in 2 cells (2%). B: DNA damage detected by localization of phosphohistone H2A.X (γ-H2AX) in nonfusion clones (NFcs) and Fcs. Cells at early passage were fixed and stained with antibodies specific for γ-H2AX and studied by immunofluorescence microscopy. Nuclei were counterstained with DAPI. C: Frequency of DNA damage. DNA damage was detected by nuclear staining for γ-H2AX in NFcs andFcs. The bars indicate means ± SD of the percentage of γ-H2AX–positive cells in nine images from three independent experiments, analyzing 156 to 360 cells in total for each clone. The number in parentheses is the modal chromosome number. D: DNA double-strand breaks detected by neutral comet assay. Cells from NFcs and Fcs were embedded in agarose, lyzed, subjected to single-cell electrophoresis, and stained with CYBR gold. Representative images are shown. E: DNA damage detected by comet assay. NFcs and Fcs were compared using comet assay. Comet tail length is expressed as units relative to tail length/mean radius of intact nuclei in nonfused cells. Fifty randomly selected cells of NFcs or Fcs were analyzed in two independent experiments. F: Relationship between binding of γ-H2AX and presence of cleaved caspase 3 (casp3) in fusion-derived cells. Cells of Fc32 were fixed and stained with antibodies specific for γ-H2AX and with antibodies specific for cleaved casp3. Three hundred γ-H2AX–positive cells were examined for cleaved casp3 staining in three independent experiments. The results indicate that few cells with bound γ-H2AX are undergoing apoptosis. G: Appearance of cells positive for γ-H2AX and cleaved casp3. The photomicrograph shows a cell typical of those positive for cleaved casp3 and diffuse γ-H2AX (arrows). Condensed chromatin probably reflects apoptosis (insets: reduced exposure of γ-H2AX and DAPI staining). Cells exhibiting speckled staining for γ-H2A, no detectable cleaved caspase 3, and fine chromatin are far more common. ∗∗∗∗P < 0.0001, one-way analysis of variance, indicating a significant difference between NFcs and Fcs.