期刊
MOLECULAR AND CELLULAR BIOCHEMISTRY
卷 333, 期 1-2, 页码 151-157出版社
SPRINGER
DOI: 10.1007/s11010-009-0215-1
关键词
Apoptosis; Caspases; Calcium signal; Thapsigargin; HL-60 cells
类别
资金
- MEC-DGI
- Junta de Extremadura [BFU2007-60091, PRI07-A024, PRE06070]
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 ([Ca2+](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 mu M UTP or 1 mu M thapsigargin induced a transient increase in [Ca2+](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 mu M 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 mu M 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 [Ca2+](c) in human myeloid HL-60 cells.
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