A. Genistein induces Top2α- and Top2β-mediated DSBs in vitro. VP-16 (0 μM (lanes 2 & 16), 3.13 μM (lanes 3 & 17), 6.25 μM (lanes 4 & 18), 12.5 μM (lanes 5 & 19), 25.0 μM (lanes 6 & 20), 50.0 μM (lanes 7 & 21), 100 μM (lanes 8 & 22)) and genistein (0 μM (lanes 2 & 16), 3.13 μM (lanes 9 & 23), 6.25 μM (lanes 10 & 24), 12.5 μM (lanes 11 & 25), 25.0 μM (lanes 12 & 26), 50.0 μM (lanes 13 & 27), 100 μM (lanes 14 & 28)) were incubated with ³²P-labeled linearized plasmid DNA in the presence of purified recombinant hTop2α (lanes 1–14) or hTop2β (lanes 15–28) as described in Materials and Methods (DNA cleavage assay). **, full length ³²P-labeled linearized plasmid DNA; *, cleaved DNA fragments. DNA cleavage products were then analyzed by agarose gel electrophoresis, followed by autoradiography. B. Genistein traps both Top2α and Top2β cleavage complexes in mouse 32Dc13 progenitor cells. The amounts of genistein-induced Top2α and the Top2β cleavage complexes were measured by the band depletion assay. 32Dc13 cells, expressing both Top2 isozymes were treated with genistein (100, 250 or 500 μM) or VP-16 (250 μM) for 15 min, followed by lysis in 6X sample buffer. Cell lysate were then analyzed by SDS-PAGE and immunoblotted with isozyme-specific antibodies. This assay measures the levels of free Top2 isozymes in the lysate as trapping on DNA leads to the decreased free Top2 levels. To demonstrate the reversibility of genistein- and VP-16-induced Top2 cleavage complexes, drug-treated cells were further incubated in drug-free medium for an additional 30 min, followed by immunoblotting analysis. C. Mouse 32Dc13 progenitor cells, expressing both Top2 isozymes, were treated with genistein (500 μM) or VP-16 (100 μM) for 0 and 2 hrs in the absence or presence of the proteasome inhibitor MG132 (10 μM). Cells were then lysed in alkaline lysis buffer followed by neutralization and S7 nuclease digestion. After SDS-PAGE, the amount of the Top2 isozymes was measured by immunoblotting.

A. Mouse 32Dc13 progenitor cells expressing control shRNA (shCtrl) or Top2β shRNA (shTop2β) were transfected with linearized pUCSV-BSD plasmid in the presence of DMSO (0.1%), VP-16 (0.5 μM) or genistein (100 μM) as indicated. Plasmid integration frequency was then determined and plotted as histograms. B. SV40-transformed Top2β^+/+ and top2β^−/− MEFs were transfected with the linearized pUCSV-BSD plasmid in the presence of DMSO (0.1%), VP-16 (0.5 μM) or genistein (10 or 20 μM) as indicated. In addition, similar transfections were also performed in the presence of the proteasome inhibitor MG132 (2 μM). Plasmid integration frequencies were then determined.

A. Primary Top2β^+/+ and top2β^−/− MEFs were treated with 25 μM CPT, 10 μM VP-16 and or increasing concentrations of genistein (25, 50, 100 μM) for 1 hour, followed by lysis in 6X SDS sample buffer and immunoblotted with antibodies against γ-H2AX and α-tubulin. B. The genistein-induced γ-H2AX signal requires proteasome activity and transcription. Primary Top2β^+/+ MEFs were treated with MG132 (10 μM) or DRB (150 μM) for 30 min, followed by co-treatment with VP-16 (10 μM), genistein (100 μM) or hydrogen peroxide (H₂O₂, 400 μM) for 1 hr. Western blotting was performed as described in A. C. Genistein-induced γ-H2AX does not require protein synthesis. Primary Top2β^+/+ MEFs were treated with cycloheximide (CHX, 10 μM) for 30 min, followed by co-treatment with VP-16 (10 μM), genistein (100 μM) or hydrogen peroxide (H₂O₂, 400 μM) for 1 hr. Western blotting was performed as described in A. D. N-acetyl cysteine had no effect on the genistein-induced DNA damage. Primary Top2β^+/+ MEFs were treated with 0.1% DMSO or 1 mM N-acetyl cysteine (NAC, 1 mM) for 30 min, followed by co-treatment with genistein (20 μM, 100 μM), VP-16 (10 μM) or H₂O₂ (100 μM, 500 μM) for 2 hrs. Western blotting was performed as described in A.

In this model, genistein stabilizes Top2β-DNA covalent adducts (Top2β cleavage complex) on chromosomal DNA within the transcribed regions. Top2β cleavage complexes arrest transcription elongation, triggering Top2β degradation through a proteasome-dependent pathway. Proteasomal degradation of Top2β cleavage complexes expose Top2β-concealed DSBs. Subsequent repair of the DSBs through the non-homologous end joining pathway (NHEJ) leads to DNA sequence rearrangements. DSBs located within MLL BCR can undergo NHEJ leading to MLL translocations and hence infant leukemias.