Research reportStaurosporine- and H-7-induced cell death in SH-SY5Y neuroblastoma cells is associated with caspase-2 and caspase-3 activation, but not with activation of the FAS/FAS-L-caspase-8 signaling pathway
Introduction
Apoptosis signal transduction and execution is associated with the coordinated action of the cascade of caspases (aspartate-specific cysteine proteases) [1], [10], [22], [38], [39]. Caspases, which are normally present as zymogens and cleaved to form active heterodimers, have the capacity to cleave aspartic acid residues. They can be divided into three subfamilies based on the preference for tetrapeptide sequences [28], [44]. Caspases act on a wide number of substrates, covering cytoskeletal and structural proteins, and proteins related with the cell cycle, DNA cleavage and DNA repair, as well as on transcription and translation factors and other transducers, protein kinases, and cytokine precursors, among others [34]. Activated caspase-3, which represents one of the ultimate steps in the caspase cascade, cleaves important nuclear and cytoskeletal proteins during apoptosis [9], [10], [34], [42], including poly(ADP-ribose) polymerase (PARP), an enzyme of 117 kDa of molecular weight involved in DNA repair [23] that is cleaved into two fragments of 89 and 28 kDa following caspase-3 activation.
The caspase cascade can be activated through independent pathways. It has been shown that mitochondrial changes, particularly those related with the release of cytochrome c, are early required steps during the process of apoptosis [13], [19], [41], [51]. Cytochrome c release from mitochondria is associated with the translocation from the cytosol to the mitochondrial membrane of the pro-apoptotic Bcl-2 family member, Bax [8], [11], [16], [41], [50], [52]. The cytosolic partner of cytochrome c in the apoptotic cascade has been identified as apoptosis protease-activating factor-1 (Apaf-1) [24], [54]. Apaf-1 and cytochrome c, in the presence of dATP, activate caspase-9 and this, in turn, activates caspase-3 [7], [14], [21], [24], [25], [54].
Another pathway of caspase activation is triggered by the Fas/Fas-L signaling system. The Fas (CD95, APO-1) receptor is a member of the death receptor subfamily of the tumor necrosis factor (TNF)/nerve growth factor (NGF) superfamily. The receptor has an extracellular domain for ligand binding and an intracellular death domain. Following binding to its specific ligand Fas-L, trimerization of Fas recruits the Fas-associated death domain (FADD) via interactions between the death domains of Fas and FADD. This is followed by FLICE/caspase-8 binding via interactions between the death-effector domains of FADD and caspase-8, and by activation of caspase-8. Activation of caspase-8, in turn, activates the caspase cascade leading to apoptosis [2], [5], [6], [12], [18], [29], [30], [32], [35], [46], [47], [49].
SH-SY5Y neuroblastoma cells have been used extensively in the study of neuronal cell death [3], [36]. Staurosporine and 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7) are non-specific protein kinase inhibitors and well-established pro-apoptotic agents [20], [31], [37], [48]. Staurosporine cell death is inhibited by Bcl-2, involves the release of cytochrome c and is associated with caspase activation [17], [19], [25], [51]. Yet the mechanisms leading to apoptosis in SH-SY5Y neuroblastoma cells following exposure to staurosporine and H-7 are not clearly understood.
The present study examines caspase activation, and more particularly the possible activation of the Fas/Fas-L-caspase-8 signaling pathway, associated with staurosporine- and H-7-induced cell death in SH-SY5Y neuroblastoma cells.
Section snippets
Materials
Staurosporine, 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7) and 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma. F12 (HAM) medium, Earle’s salts base (MEM-EAGLE), trypsin–EDTA, and fetal bovine serum were obtained from Biological Industries.
Cell cultures and experimental treatments
SH-SY5Y human neuroblastoma cells were grown on 6-well plates to confluency at 37°C with 5% CO2 in a humidified atmosphere with Dulbecco’s modified Eagle’s medium and Nutrient Mixture F-12 with l-glutamine
Induction of cell death in SH-SY5Y cells by staurosporine and H-7
Following staurosporine (100 nM) or H-7 (100 μM), SH-SY5Y cells became rounded and were loosely attached to the plate. Under phase contrast microscopical optics, dying cells exhibited morphological characteristics of apoptosis, including extreme nuclear shrinkage and chromatin condensation, as well as apoptotic bodies (Fig. 1). Dying cells were observed at 3 h and increased in number at 6 and 12 h. Similar morphological signs of apoptosis were seen in cells stained with propidium iodide (data
Discussion
The present results support the idea that staurosporine and H-7 induce cell death in SH-SY5Y human neuroblastoma cells, and that this process is associated with caspase activation [4], [25], [27]. The process is accompanied by the formation of specific PARP-cleaved products of 89 kDa whose appearance is blocked through DEVD-fmk, an irreversible caspase-3 (and caspase-8) inhibitor. Previous reports have shown that staurosporine-induced cell death in SH-SY5Y neuroblastoma cells is accompanied by
Acknowledgements
This work was supported in part by FIS grant 99-1118 and EC contract QLG3-CT-1999-602. We wish to thank T. Yohannan for editorial assistance.
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