Caspase-3 and -9 are activated in human myeloid HL-60 cells by calcium signal.

Abstract

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.

• Authors: D González, J Espino, I Bejarano, J J López, A B Rodríguez, J A Pariente
• Journal: Molecular and cellular biochemistry (Mol Cell Biochem) Vol. 333 Issue 1-2 Pg. 151-7 (Jan 2010) ISSN: 1573-4919 [Electronic] Netherlands
• PMID: 19626422 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Thapsigargin
• Caspase 3
• Caspase 9
• Calcium
• Uridine Triphosphate

Topics

• Calcium (analysis)
• Calcium Signaling (physiology)
• Caspase 3 (analysis, metabolism)
• Caspase 9 (analysis, metabolism)
• Cytosol (chemistry)
• HL-60 Cells
• Humans
• Kinetics
• Myeloid Cells (metabolism)
• Thapsigargin (pharmacology)
• Uridine Triphosphate (pharmacology)