Objectives: 

In the present study, the authors investigated whether intracellular reactive oxygen species (ROS) generation plays a role on tumor necrosis factor alha (TNFα)-induced apoptosis in human histiocytic lymphoma cell line U937.

Materials: 

Apoptosis was quantified using a fluorescence microscope staining with 10 μg/mL Hoechst 33342. We have also analysed mitochondrial membrane depolarisation using MitoTracker Red (MTR) and intracellular ROS production was determined with two different dyes: 2',7'-dichlorofluorescin diacetate (H2DCFDA) and dihydrorhodamine 123 (DHR). Analyses were performed by flow cytometry. Statistical analysis were performed using Wilcoxon's test between groups, and Friedman's test when comparisons of different times of the same treatment were considered.

Results: 

The maximal apoptosis percentage in U937 cells treated with 10 ng/mL TNF&#945; in presence of 1 &#956;M cycloheximide (CHX), an inhibitor of protein biosynthesis, was after 3.5 hours of treatment, which was significantly higher (p < 0.05) than non-treated cells. Our results also show that treatment of cells with TNF&#945; plus CHX induced a significative increase in intracellular ROS generation. The stimulatory effect of TNF&#945; on oxidative signal was followed by a mitochondrial membrane depolarisation. Pretreatment of cells with the water soluble derivative of vitamin E trolox (100 mM) for 30 min, or 1 mM N-acetyl cysteine (NAC) for 1 hour, significantly reduced intracellular ROS generation, mitochondrial depolarization and percentage of apoptosis produced by TNF&#945; plus CHX.

Conclusions: 

Our results suggest that TNF&#945;-induced apoptosis in human human histiocytic lymphoma cell line U937 is dependent of intracellular ROS generation.