Aim: 

In the present study we have evaluated the effect of hydrogen peroxide (H₂O₂) on apoptosis in human leukemia cell line HL-60.

Methods: 

Caspase-3 and -9 activities were determined from the cleavage of the respective specific fluorogenic substrates and measured with a fluorescence spectrophotometer. The permeability transition pore (PTP) induction was determined with calcein in the presence of cobalt chloride to quench calcein fluorescence from all cellular domains except within mitochondria. Apoptosis and/or necrosis, and the cell cycle phase distribution were analysed by flow cytometry using annexin V-FITC and propidium iodide. The amount of proapoptotic protein activated was determined by immunoprecipitation and western blotting using specific antibody directed towards the active form of Bid.

Results: 

Our results show that treatment of cells with H₂O₂ resulted in a time- and concentration-dependent increase in caspase-3 and -9 activation, reaching maximal caspase activities after 120 min of stimulation at 100 mM. Aditionally, H₂O₂ also induced upregulation of proapoptotic members, such as Bid, and PTP induction, which was reflected by a loss of mitochondrial staining by calcein. Finally, after 120 min of incubation with 100 mM H₂O₂ the intensity of annexin V and propidium iodide fluorescence increased in comparison with control cells. The effect of H₂O₂ was similar compared to that obtained with 1 mM thapsigargin for 1 hour.

Conclusion: 

Our results suggest that H₂O₂ has proapoptotic effects on the human myeloid cell line HL-60.

Supported by MEC-DGI grant BFU2007-60091. P.C. Redondo was supported by MEC-Ramón y Cajal program (RYC2007-00349).