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

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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 ([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 μM UTP or 1 μM 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 μM 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 μM 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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Abbreviations

BAPTA-AM:

1,2-bis(o-aminophenoxy)ethane-N,N,N′,N-tetraacetic acid-acetoxymethyl ester

[Ca2+]c :

Cytosolic free calcium concentration

CHAPS:

3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate

DMSO:

Dimethylsulfoxide

DTT:

Dithiothreitol

EGTA:

Ethylene glycol-bis (2-aminoethyleter)-N,N,N′,N′-tetraacetic acid

ER/SR:

Sarco-endoplasmic reticulum

FCCP:

Carbonyl cyanide 4-(trifluoromethoxy)phenyl-hydrazone

HEPES:

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

IP3 :

Inositol 1,4,5-triphosphate

PLC:

Phospholipase C

ROS:

Reactive oxygen species

SERCA:

Sarco-endoplasmic reticulum Ca2+-ATPase

TG:

Thapsigargin

UTP:

Uridine-5′-triphosphate

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Acknowledgments

This work was supported by MEC-DGI and Junta de Extremadura grants BFU2007-60091 and PRI07-A024, respectively. I. Bejarano was beneficiary of grant by Junta de Extremadura PRE06070.

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Correspondence to J. A. Pariente.

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González, D., Espino, J., Bejarano, I. et al. Caspase-3 and -9 are activated in human myeloid HL-60 cells by calcium signal. Mol Cell Biochem 333, 151–157 (2010). https://doi.org/10.1007/s11010-009-0215-1

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  • DOI: https://doi.org/10.1007/s11010-009-0215-1

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