TPA-induced differentiation sensitizes U937 cells to arsenic trioxide (As₂O₃)-induced differentiation.

(A) Undifferentiated (▴), TPA-differentiated (●), and Bcl-2 overexpressing U937 cells (▪, U-BCL2) were treated with increasing concentrations of As₂O₃ for 72 hours (left panel) or with 4 μM As₂O₃ for indicated times (right panel). Apoptotic DNA fragmentation was quantified with the use of a filter elution assay. Values from 1 of 2 independent experiments are shown (mean ± SD of triplicate samples). (B) Western blot analysis of cytochrome c and Smac/Diablo expression in cytosolic fractions from undifferentiated (U937) and TPA-differentiated (U-TPA) U937 cells and U937 cells overexpressing Bcl-2 (U-BCL2) treated with 4 μM As₂O₃ for indicated times. (C) Mitochondrial membrane depolarization was visualized by flow cytometry in U937, U-TPA, and U-BCL2 cells treated with 4 μM As₂O₃ for indicated times. Mitochondrial depolarization was identified by an increased level of cytosolic green monomer (FL1-H; dashed lines indicate the controls). (D) Western blot analysis of procaspase-3 and caspase-3 active fragments and PARP expression in whole-cell extracts from U937, U-TPA, and U-BCL2 cells treated with 4 μM As₂O₃ for indicated times. Numbers are molecular weights in kilodaltons. *Cleavage products. Loading was checked with the use of an antihuman HSC70 mAb.