Characterization of CPP32‐Like Protease Activity Following Apoptotic Challenge in SH‐SY5Y Neuroblastoma Cells

Abstract

We characterized the activation of interleukin‐1β‐converting enzyme (ICE)‐like proteases (caspases) in human neuroblastoma cells (SH‐SY5Y) following challenge with staurosporine, an established agent known to induce apoptosis. Time course analyses of lactate dehydrogenase release detected a significant increase in cell death as early as 6 h that continued at least until 24 h following staurosporine treatment. Western blot analyses using anti‐poly(ADP‐ribose) polymerase (anti‐PARP) and anti‐CPP32 antibodies revealed proteolytic processing of CPP32 (an ICE homologue) as well as fragmentation of PARP as early as 3 h following staurosporine challenge. Furthermore, the hydrolysis of the CPP32 substrate acetyl‐DEVD‐7‐amido‐4‐methylcoumarin was detected as early as 3 h and became maximal at 6 h after staurosporine challenge, suggesting a delayed and sustained period of CPP32‐like activation. In addition, we used the first immunohistochemical examination of CPP32 and PARP in cells following an apoptotic challenge. The localization of CPP32 in untreated SH‐SY5Y cells was exclusively restricted to the cytoplasm. Following staurosporine challenge there was a condensing of CPP32 immunofluorescence from the cytoplasm to a region adjacent to the plasma membrane. In contrast, PARP immunofluorescence was evenly distributed in the nucleus in untreated SH‐SY5Y cells and on staurosporine challenge was found to be associated with condensed chromatin. It is important that a pan ICE inhibitor [carbobenzoxy‐Asp‐CH2OC(O)‐2,6‐dichlorobenzene] was able to attenuate lactate dehydrogenase release and PARP and CPP32 cleavage and altered immunohistochemical staining patterns for PARP and CPP32 following staurosporine challenge.

Alterations of Extracellular Calcium Elicit Selective Modes of Cell Death and Protease Activation in SH‐SY5Y Human Neuroblastoma Cells

“…Inhibition of caspase-3 activity attenuates apoptosis in many mammalian cell types including neuronal cells (Nath et al, 1 9 9 6~;Posmantur et al, 1997). After apoptotic injury, the 32-kDa caspase-3 proenzyme is cleaved to 17-and 12-kDa fragments, which form the active heterodimer (Thornberry et al, 1992;Walker et al, 1994).…”

“…Caspase-3-like activity was directly examined in cell lysates from EGTA-or Ca*+,-treated SH-SY5Y cells. Activity was measured at 0, 1, 2, 4, and 8 h after treatment, using the fluorogenic caspase-3-like protease substrate Ac-DEVD-MCA (Nath et al, 19966;Posmantur et al, 1997). An increase in caspase-3-like activity is apparent 2 h after treatment but is not significant until 4 h after start of treatment (5.56 2 0.98 fluorescent U/pg of protein for control vs. 30.9 2 1.8 for EGTA treated).…”

“…We have previously demonstrated the activation of two families of cysteine proteases, caspases and calpains, in SH-SY5Y cells undergoing staurosporineinduced apoptosis Posmantur et al, 1997). Caspase-3 has been studied as a putative mediator of mammalian apoptosis (Fernandes-Alnemri et al, 1994;Casciola-Rosen et al, 1996;Posmantur et al, Alterations in intracellular calcium (Ca2+,) homeostasis have been implicated in the onset of cell death in many cell types. However, the mode of cell death and direction of change in Ca2fi differ among cell types McConkey and Orrenius, 1997).…”

Biological Role of Anaplastic Lymphoma Kinase in Neuroblastoma

“…21 High expression of caspase-1, -3, and -8 is correlated with favorable neuroblastomas. 22,23 On the other hand, survivin, which suppresses caspase and promotes the cell survival signal, is significantly expressed, 24 and telomerase is activated 25 in unfavorable tumors. There may be a critical difference in the expression of other molecules, including RTKs, in neuroblastoma.…”

Evidence for Activation of Caspase‐3‐Like Protease in Excitotoxin‐ and Hypoxia/Hypoglycemia‐Injured Neurons

“…For example, caspase-3 (previously called CPP32, Yama, or apopain) is found to be almost universally activated in numerous cell types when subjected to various apoptotic challenges. Three biochemical events are observed during caspase-3 activation: (a) processing of intact 32-kDa caspase-3 to the 17-and 12-kDa dimeric form (Nicholson et al, 1995;Posmantur et al, 1997), (b) fragmentation of the 11 3-kDa poly (ADP-nibose) polymerase (PARP) to an 89-kDa form (Lazebnik et al, 1994), and (c) processing of nonerythroid cr-spectrin (a-spectrin; 280 kDa) into a 120-kDa spectrin breakdown product (SBDP12O) (Martin et al, 1995). In fact, recently caspase-3, PARP, and a-spectrin processing have been reported in several neuronal apoptosis models, such as cultured neuroblastoma or neuronal cells exposed to staurosporine as well as cerebellar granule neurons subjected to potassium deprivation Nath et al, 1996a;Wiesner and Dawson, 1996;Armstrong et al, 1997;Ni et al, 1997).…”