Fig. 9.

MG132 inhibits the reversal of etoposide-induced TOP2A- and TOP2B-DNA complexes. (A) K562 cells were incubated with solvent, etoposide (100 μM), MG132 (50 μM) or were co-incubated with 50 μM MG132 and 100 μM etoposide for 2 h. After 2 h etoposide was removed, but MG132 was maintained in cell incubations that initially contained it. Levels of TOP2A and TOP2B DNA complexes at 0, 15, 30, 60 and 120 min after etoposide removal (wash-out) were determined using the TARDIS assay. Statistical comparisons were made between the levels of TOP2-DNA complexes in the presence or absence of MG132 by unpaired t-test. Figures are given below for cell viability (trypan blue exclusion) under the cell treatment conditions used: “−120 min” refers to the time at which drugs were first added to cells, “0” refers to the time at which etoposide wash-out was performed and “120 min” refers to 2 h post drug wash-out. (B) The treatments employed in (A) do not significantly affect cellular TOP2A or TOP2B levels. Cells were treated as indicated and fixed with paraformaldehyde on poly lysine-coated slides. Total TOP2A and TOP2B was quantified by immunofluorescence. 100 μM etoposide incubation did result in a small decrease in total cellular TOP2A. (C) Cell cycle distribution at time of etoposide wash-out (“0”) and 2 h after etoposide washout (“120”).