Aim: 

In the present study, the authors investigated whether cytosolic free calcium concentration ([Ca²⁺]_c) plays a role in hydrogen peroxide (H₂O₂)-induced caspase activation in human leukemia cell line HL-60.

Methods: 

We have analysed mitochondrial membrane depolarisation, [Ca²⁺]_c determination and caspase-3 and -9 activities by fluorimetric methods, using the fluorescent dye tetramethylrhodamine (TMRM) and Fura-2, and specific fluorogenic substrates, respectively.

Results: 

Our results show that treatment of cells with H₂O₂ (1 mM) induced a transient increase in [Ca²⁺]_c due to calcium release from internal stores. The stimulatory effect of H₂O₂ on calcium signal was followed by a mitochondrial membrane depolarisation. Similar effects both on [Ca²⁺]_c and mitochondrial depolarisation were obtained when the cells were treated with the calcium mobilising agonist UTP (10 mM) or the specific inhibitor of calcium reuptake thapsigargin (1 mM). Loading of cells with 10 mM dimethyl BAPTA, an intracellular Ca²⁺ chelator, for 30 min significantly reduced both H₂O₂-, UTP- or thapsigargin-induced mitochondrial depolarisation and caspase activation. Similar results were obtained when the cells were pretreated with 10 mM Ru360 for 30 min, a specific blocker of calcium uptake into mitochondria.

Conclusion: 

Our results suggest that H₂O₂-induced caspase-3 and -9 activation in human myeloid cell line HL-60 is dependent on calcium signalling.

Supported by MEC-DGI grant BFU2007-60091. D. González was supported by University of Extremadura grant