Protein kinase inhibitor, staurosporine, induces a mature neuronal phenotype in SH‐SY5Y human neuroblastoma cells through an α‐, β‐, and ζ‐protein kinase C‐independent pathway

Abstract

Previous studies have shown that the tumour-promoting phorbol ester 12-O-tetradecanoyl phorbol-13 acetate (TPA) induces both morphological and functional differentiation in SH-SY5Y human neuroblastoma cells (Påhlman et al., 1981). In order to investigate the role of protein kinase C (PKC) in TPA-induced maturation of SH-SY5Y cells, we have used staurosporine, which is a potent inhibitor of protein kinases including PKC. Treatment of SH-SY5Y cells with 25 nM staurosporine for 72 hours caused an appearance of long, neuritelike processes with varicosities, terminated by growth cones. The morphological differentiation was accompanied by a cessation of DNA synthesis, induction of growth associated protein 43 (GAP-43), and neuropeptide Y (NPY) mRNA. These effects of staurosporine were comparable to those elicited by TPA. Staurosporine further induced a time-dependent increase in the expression of tyrosine hydroxylase protein and a 30-fold increase in the concentration of noradrenaline. TPA only induced a marginal increase in tyrosine hydroxylase expression. Both TPA and staurosporine induced an appearance of voltage-gated Ca2+ channels in SH-SY5Y cells detected with single-cell fluorescent measurements using fura-2. The Ca2+ channels were found almost exclusively in growth cones and varicosities. Staurosporine inhibited both basal and a TPA-induced phosphorylation of an endogenous 80kDa PKC substrate (p80), and also blocked c-fos proto-oncogene mRNA expression induced by the phorbol ester. Bryostatin 1, a potent activator of PKC, has failed to induce morphological or functional differentiation in SH-SY5Y cells (Jalava et al., 1990). Incubation of SH-SY5Y cells in the presence of 100 nM bryostatin 1 for 24 hours caused a complete disappearance of all immunoreactive alpha-, beta-, and zeta-PKC. The level of epsilon-PKC decreased by 70%. Staurosporine induced a partial translocation of the epsilon-isoenzyme but it failed to cause down-regulation of epsilon-PKC. Bryostatin 1-treatment did not interfere in the ability of staurosporine to induce morphological differentiation, cessation of DNA synthesis, and GAP-43 and NPY mRNA expression. The ability of staurosporine to stimulate tyrosine hydroxylase expression and to increase cellular content of noradrenaline was also unaffected. Taken together the results of this study show that staurosporine induces a mature neuronal noradrenergic phenotype in SH-SY5Y cells through an alpha-, beta-, and zeta-PKC-independent pathway.

Translocation and Phosphorylation of Calcyclin Binding Protein during Retinoic Acid-induced Neuronal Differentiation of Neuroblastoma SH-SY5Y Cells

“…There are many examples in literature of the distinct cell types whose differentiation is under the control of RA. SH-SY5Y cells, a neuroblastic subclone of the neuroblastoma cell line SK-N-SH, withdraw from the cell cycle and exhibit a distinct neuronal phenotype when treated with different agents, such as neuortrophic factors (Kaplan et al, 1993), retinoic acid (Kaplan et al, 1993), phorbol ester (Pahlman et al, 1981), or staurosporine (Jalava et al, 1992;Jalava et al, 1993). We first cloned the human CacyBP gene during the differentiation of the gliomablastoma cell, BT325.…”

Differential responses of oligodendrocytes to tumor necrosis factor and other pro-apoptotic agents: Role of ceramide in apoptosis

“…Drugs such as staurosporine (Jalava et al, 1993;Raff et al, 1993;Mangoura and Dawson, 1998) and inhibitors of the PtdIns 3-kinase pathway (e.g., wortmannin and the structurally unrelated LY294002; Yao and Cooper, 1995), also induce apoptosis in many cells, including oligodendrocytes (Arcaro and Wymann, 1993;Straub and Sharp, 1996;Vemuri and McMorris, 1996). In several preparations of neuronal and glial cells, we have shown that such drug-induced apoptosis is associated with the increased formation of ceramide Dawson, 1996a,b, 1997;Dawson et al, 1997;Mangoura and Dawson, 1998), suggesting that common pathways may be shared with TNF-␣-induced apoptosis.…”

Tissue Transglutaminase Mediates Activation of RhoA and MAP Kinase Pathways during Retinoic Acid-induced Neuronal Differentiation of SH-SY5Y Cells

“…Many studies on a variety of embryogenic neuronal cell types have shown that RA can stimulate both neurite number and length (4) and neurite outgrowth in amphibian spinal cord (5). SH-SY5Y cells, a neuroblastic subclone of the neuroblastoma cell line SK-N-SH, withdraw from the cell cycle and exhibit a distinct neuronal phenotype when treated with different agents such as neurotrophic factors (6), retinoic acid (6), phorbol ester (7), or staurosporine (8,9). The RA-induced change to a neuronal phenotype in SH-SY5Y cells is associated with increased expression of tissue transglutaminase (TGase) (10 -12).…”