Figure 1.
MLL rearrangement after etoposide treatment.
(A) Schematic diagram of etoposide (VP) or Bleomycin (BLM) exposure and subsequent culture for recovery. (B-D) Dual-color FISH analysis of AT5BIVA and 11-4 cells, cultured for 30 min (w30) and 6 hours (w6h) after etoposide exposure (Con; unexposed cells). The cells were hybridized with paired probes spanning the MLL gene with overlap in the BCR (centromeric side in green, telomeric side in red). (B) The representative images of FISH (BIVA w6h) are shown. Arrowheads indicate the split signals (separated >1 µm). Scale bar: 5 µm. (C) The percentage of cells with split signals was significantly increased for AT5BIVA cells or the ATM kinase inhibitor treated 11-4 cells compared to normal 11-4 cells cultured for 30 min (w30) or 6 hours (w6h) in normal medium after etoposide treatment (*P<0.05, **P<0.0001 as determined by the Z test of homogeneity for independent samples, respectively). (D) 11-4 cells were transfected with a RAD51 expression vector (RAD51) or an empty vector (mock). Twenty two hours after transfection, cells were treated with etoposide and cultured for 30 min (w30) and 6 hours (w6h) in normal medium (Con; unexposed cells). The percentage of cells with split signals was significantly higher in RAD51 overexpressing 11-4 cells compared to empty vector transfected cells after culturing for 30 min and 6 hours in normal medium (*P<0.01, **P<0.0001 as determined by the Z test).
Figure 2.
Focus formation and expression of RAD51 in ATM deficient cells.
(A) Immunoblotting analysis of BV173, AT5BIVA and 11-4 cells using an anti-RAD51 antibody. Cells were treated with etoposide (w30) or DMSO (Con) for 10 min and cultured for 30 min in normal medium. β-actin was used as a loading control. (B) The percentage of RAD51 foci positive AT5BIVA, 11-4 and BV173 cells was significantly increased after etoposide or bleomycin treatment (p<0.001, as determined by the Z test of homogeneity for independent samples). Values represent the means ± SE from three to four independent experiments (n>200 cells for each). (C) Focus formation of RAD51 in AT5BIVA and 11-4 cells after etoposide exposure. RAD51 nuclear foci were detected by immunofluorescence staining using anti-RAD51 antibodies. Scale bar: 5 µm.
Figure 3.
Binding of RAD51 to breakpoint clustering region in the MLL gene after etoposide treatment.
(A) Schematic representation of the BCR in the MLL gene. The arrow indicates the translocation breakpoint hotspot identified in treatment-related leukemias. Asterisks represent topoisomerase II consensus sites. Positions of primer sets used in real-time PCR analysis (t2, t4, bbt56, bt56, t56 and in14) are indicated. (B-D) ChIP analysis of BV173 (B), 11-4 (C) and AT5BIVA (D) cells was performed with anti-RAD51 antibodies, immediately after etoposide (VP), or vehicle (Con) exposure, and 30 min after recovery in normal medium (w30). DNA was analyzed by real-time PCR using the primers indicated in (A) and that for a control region in the β-globin gene (βG). Values represent the means ± SE from five independent experiments. *: P<0.05 (compared to βG by Student's t test).
Figure 4.
RPA is loaded on the BCR after etoposide treatment.
(A, B) ChIP analysis after etoposide treatment of 11-4 (A) and AT5BIVA (B) cells using antibodies against RPA was performed as described in Figure 3. Values represent the means ± SE from three independent experiments. *P<0.05 (compare to βG by Student's t test). (C) Immunoblotting analysis of AT5BIVA and 11-4 cells using anti-RPA32 antibodies. Cells were treated with etoposide for 10 min (VP) and allowed to recover in normal medium for 30 min (w30) or 2 hours (w2h). Slower migrating bands, indicated as RPA32-P, in the long exposed blot were lost by λ-phosphatase (λPPase) treatment. β-actin was used as a loading control.
Figure 5.
INO80 loading on the BCR after etoposide treatment.
(A-C) ChIP analysis of BV173 (A), 11-4 (B) and AT5BIVA (C) cells after etoposide exposure was performed with antibodies against INO80 as described in Figure 3. Values are the means ± SE from three independent experiments. (D) Immunoblotting analysis of BV173, AT5BIVA and 11-4 cells was performed using anti-INO80 antibodies. RAD21 was used as a loading control. * P<0.05 (compared to βG by Student's t test).