Inhibition of etoposide-induced apoptosis in U937 cells induced to differentiate by TPA.

U937 cells were untreated (TE) or preincubated for indicated times with 20 nM TPA, washed, then treated for 4.5 hours with 50 μM etoposide. (A) Nuclear morphologic changes associated with apoptosis were visualized by fluorescent microscopy after Hoechst staining. (B) (upper panel) Apoptotic DNA fragmentation was quantified using a filter elution assay. Values from 1 of 2 independent experiments are shown (mean ± SD of triplicate samples). (inset) Visualization of apoptotic DNA fragmentation by agarose gel electrophoresis. (lower panel) Percentage of differentiated cells according to CD11b expression measured before etoposide treatment. (C) Indicated caspases and PARP were analyzed in whole-cell extracts by immunoblot. Numbers are molecular weights in kilodaltons. “Procaspase” and “caspase” are used to design the studied enzymes, depending on whether the tested antibody does not or does detect the cleavage fragments (∗), respectively. A unique series of cell lysates was used for these experiments. Loading of each strip was checked by using an antihuman HSC70 mAb (one representative control is shown).