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. 2011 Aug 1;286(38):33591–33600. doi: 10.1074/jbc.M111.258137

FIGURE 5.

FIGURE 5.

ITCs induce reversible Top2 cleavage complexes in vitro. Left panels (A–C), ITCs induce DNA cleavage in the presence of purified hTop2α. DNA cleavage assay was performed as described in under “Experimental Procedures” using purified recombinant hTop2α. The concentrations of VM-26, VP-16, and disulfiram were 5, 20, and 100 μm, respectively. The concentrations of PEITC (A, left panel) and BITC (B, left panel) were from 0.01 μm to 10 mm with a 10-fold serial dilution, whereas concentrations of SFN (C, left panel) were from 1 μm to 10 mm. DNA cleavage was measured after a 30-min incubation. Right panels (A–C), post-reaction EDTA treatment reverses ITC-induced DNA cleavage. To test the reversibility of ITC-induced DNA cleavage, 50 mm EDTA (final concentration) was added to each cleavage reaction after a 30-min preincubation with PEITC (1 mm) (A), BITC (1 mm) (B), and SFN (10 mm) (C). D, ITC-induced Top2 cleavage complex is greatly reduced in the presence of glutathione (GSH). DNA cleavage was performed as described except that increasing concentrations of GSH (5 μm to 0.5 mm with 10-fold increments) was co-incubated with VP-16 (20 μm), disulfiram (100 μm), or BITC (100 μm). DNA cleavage products of all reactions were analyzed by 1% agarose gel electrophoresis followed by autoradiography. Organo-selenium induce reversible Top2 cleavage complexes in vitro. Left panels (E and F), organo-selenium induce DNA cleavage in the presence of purified hTop2α. DNA cleavage assay was performed as described under “Experimental Procedures” using purified recombinant hTop2α. The concentrations of VM-26, VP-16, and disulfiram were 5, 20, and 100 μm, respectively. The concentrations of selenocysteine (E, left panel) were from 0.1 μm to 10 mm with a 10-fold serial dilution. The concentrations of selenomethionine (F, left panel) were from 1 μm to 10 mm with a 10-fold serial dilution. DNA cleavage was measured after a 30-min incubation. Right panels (E and F), post-reaction EDTA treatment reverses ITC-induced DNA cleavage. To test the reversibility of organo-selenium-induced DNA cleavage, 50 mm EDTA (final concentration) was added to each cleavage reaction after a 30-min preincubation with selenocysteine (E; 10 mm), selenomethionine (F, 10 mm). After another 30 min, DNA cleavage was then measured. As controls, VM-26 (5 μm) or disulfiram (100 μm) were also included.