Caspase-3 and -9 are activated in human myeloid HL-60 cells by calcium signal.
This study is aimed to determine the role of calcium signaling evoked by the calcium-mobilizing agonist uridine-5'-triphosphate (UTP) and by the specific inhibitor of the endoplasmic reticulum calcium reuptake thapsigargin on caspase activation in human leukemia cell line HL-60. We have analyzed cytosolic free calcium concentration ([Ca(2+)](c)) determination, mitochondrial membrane potential and caspase-3 and -9 activity by fluorimetric methods, using the fluorescent ratiometric calcium indicator Fura-2, the dye JC-1, and specific fluorogenic substrate, respectively. Our results indicated that treatment of HL-60 cells with 10 microM UTP or 1 microM thapsigargin induced a transient increase in [Ca(2+)](c) due to calcium release from internal stores. The stimulatory effect of UTP and thapsigargin on calcium signal was followed by a mitochondrial membrane depolarization. Our results also indicated that UTP and thapsigargin were able to increase the caspase-3 and -9 activities. The effect of UTP and thapsigargin on caspase activation was time dependent, reaching a maximal caspase activity after 60 min of stimulation. Loading of cells with 10 microM dimethyl BAPTA, an intracellular calcium chelator, for 30 min significantly reduced both UTP- or thapsigargin-induced mitochondrial depolarization and caspase activation. Similar results were obtained when the cells were pretreated with 10 microM Ru360 for 30 min, a specific blocker of calcium uptake into mitochondria. The findings suggest that UTP- and thapsigargin-induced caspase-3 and -9 activation and mitochondrial membrane depolarization is dependent on rises in [Ca(2+)](c) in human myeloid HL-60 cells.
González
D
D
Department of Physiology, Faculty of Science, University of Extremadura, 06071, Badajoz, Spain.
Espino
J
J
Bejarano
I
I
López
J J
JJ
Rodríguez
A B
AB
Pariente
J A
JA
D002118:Calcium / Q000032:analysis; D020013:Calcium Signaling / Q000502:physiology*; D053148:Caspase 3 / Q000032:analysis / Q000378:metabolism*; D053453:Caspase 9 / Q000032:analysis / Q000378:metabolism*; D003600:Cytosol / Q000737:chemistry; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D022423:Myeloid Cells / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D014544:Uridine Triphosphate / Q000494:pharmacology
D019284:Thapsigargin; D053148:Caspase 3; D053453:Caspase 9; D002118:Calcium; D014544:Uridine Triphosphate