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. 2009 Jun 26;10(8):887–893. doi: 10.1038/embor.2009.97

Figure 3.

Figure 3

Transcription-induced DNA double-strand breaks and ataxia telangiectasia mutated activation in post-mitotic cells in response to topoisomerase 1 cleavage complexes. (A,B) Inhibition of CPT-induced γ-H2AX by transcription inhibitors in human lymphocytes. Cells were treated for (A) 1 h with DRB (100 μM) or FLV (1 μM) or (B) for 17 h with α-amanitin (10 μM) before the addition of CPT (2 h with the indicated concentrations in (A); 2 h with 25 μM in (B). The percentage of γ-H2AX-positive cells (average±standard deviation) were determined by flow cytometry as described in Fig 1B. Asterisks in (B) denote significant difference from CPT-treated cells in the absence of α-amanitin (P<0.001; t-test). (C) FLV prevents CPT-induced γ-H2AX foci in rat neurons. Cells were treated with FLV (1 μM, 1 h) before the addition of CPT (25 μM, 1 h) and then stained for γ-H2AX (green). DNA was counterstained with PI (red). Scale bar, 5 μm. (D) Quantification of γ-H2AX and ATM-PS1981 signal intensity per nucleus (average±standard deviation) in rat cortical neurons treated as described in (C) and (G). (E,F) FLV prevents CPT-induced DSBs in rat neurons. Cells were treated with FLV (1 μM, 1 h) before the addition of CPT (25 μM, 1 h) and DSBs were detected by neutral COMET assay. (E) Representative picture of nuclei. (F) Quantification of the COMET tail length (average±standard deviation, 30–40 cells were examined per group). Asterisks denote significant difference from CPT-treated cells in the absence of FLV (P<0.001; t-test). (G) FLV prevents CPT-induced ATM-PS1981 in rat neurons. Cells were treated with FLV (1 μM, 1 h) before the addition of CPT (25 μM, 1 h) and then stained for ATM-PS1981 (green). DNA was counterstained with PI (red). Scale bar, 5 μm. (H) FLV prevents CPT-induced ATM-PS1981 in human lymphocytes. Western blotting of ATM-PS1981 and total ATM in cells treated with FLV (1 μM, 1 h) before the addition of CPT (25 μM, 1 h). ATM, ataxia telangiectasia mutated; ATM-PS1981, phosphorylation of ATM on Ser 1981; CPT, camptothecin; DRB, 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside; DSBs, double-strand breaks; FLV, flavopiridol; γ-H2AX, phosphorylated histone H2AX; PI, propidium iodide.