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 (original) (raw)

1993, Journal of Cellular Physiology

Previous studies have shown that the tumour-promoting phorbol ester 12-0tetradecanoyl phorbol-13 acetate (TPA) induces both morphological and functional differentiation in SH-SY5Y human neuroblastorna cells (Pshlrnan 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 n M 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) rnRNA. 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 SO-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 Ca'+ channels in SH-SY5Y cells detected with single-cell fluorescent measurements using fura-2. The Ca'+ 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 rnRNA 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 n M bryostatin 1 for 24 hours caused a complete disappearance of all irnrnunoreactive a-, p-, and 5-PKC. The level of E-PKC decreased by 70%. Staurosporine induced a partial translocation of the &-isoenzyme but it failed to cause down-regulation of E-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 a-, p-, and 5-PKC-independent pathway. o 1993 WiIey-Liss, Inc. SH-SY5Y human neuroblastoma cells are induced t o differentiate t o m a t u r e n e u r o n a l cells w h e n treated with tumour-promoting p h o r b o l esters such as TPA (for a review, see PBhlman e t al., 1990). Characteristic t o TPA-treated S H-S Y 5 Y cells i s an appearance o f long, n e u r i t e l i k e processes with varicosities and g r o w t h cones (PBhlman e t al., 1981), an increased expression o f mRNA for axon-specific g r o w t h associated p r o t e i n (GAP-43) (Bjelfman e t al., 1990), elevated concentrations o f noradrenaline and neuron-specific enolase (Pbhlman e t al., 1981), and an appearance of voltage gated Ca2+ channels (H e i k k i l a e t al., 1989). S H-S Y 5 Y 0 1993 WILEY-LISS, INC cell differentiation i s also associated with a down-regul a t i o n o f c-myc proto-oncogene mRNA (H a m m e r l i n g e t al., 1987; Jalava et al., 1988) and p r o t e i n expression (Jalava e t al., 1992) as w e l l as an increased pp6OC-"'" kinase a c t i v i t y (Bjelfman e t al., 1990).