PROTEIN-KINASE INHIBITOR, STAUROSPORINE, INDUCES A MATURE NEURONAL PHENOTYPE IN SH-SY5Y HUMAN NEUROBLASTOMA-CELLS THROUGH AN ALPHA-PROTEIN,BETA-PROTEIN, AND ZETA-PROTEIN KINASE C-INDEPENDENT PATHWAY

Previous studies have shown that the tumour-promoting phorbol ester 12 -O-tetradecanoyl phorbol-13 acetate (TPA) induces both morphological a nd functional differentiation in SH-SY5Y human neuroblastoma cells (Pa hlman et al., 1981). In order to investigate the role of protein kinas e C (PKC) in TPA-induced maturation of SH-SY5Y cells, we have used sta urosporine, which is a potent inhibitor of protein kinases including P KC. Treatment of SH-SY5Y cells with 25 nM staurosporine for 72 hours c aused an appearance of long, neuritelike processes with varicosities, terminated by growth cones. The morphological differentiation was acco mpanied by a cessation of DNA synthesis, induction of growth associate d 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 tyrosi ne hydroxylase protein and a 30-fold increase in the concentration of noradrenaline. TPA only induced a marginal increase in tyrosine hydrox ylase 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 a lmost exclusively in growth cones and varicosities. Staurosporine inhi bited both basal and a TPA-induced phosphorylation of an endogenous 80 kDa PKC substrate (p80), and also blocked c-fos proto-oncogene mRNA ex pression induced by the phorbol ester. Bryostatin 1, a potent activato r of PKC, has failed to induce morphological or functional differentia tion in SH-SY5Y cells (Jalava et al., 1990). Incubation of SH-SY5Y cel ls in the presence of 100 nM bryostatin 1 for 24 hours caused a comple te disappearance of all immunoreactive alpha-, beta-, and zeta-PKC. Th e level of epsilon-PKC decreased by 70%. Staurosporine induced a parti al translocation of the E-isoenzyme but it failed to cause down-regula tion of E-PKC. Bryostatin 1-treatment did not interfere in the ability of staurosporine to induce morphological differentiation, cessation o f DNA synthesis, and GAP-43 and NPY mRNA expression. The ability of st aurosporine to stimulate tyrosine hydroxylase expression and to increa se cellular content of noradrenaline was also unaffected. Taken togeth er the results of this study show that staurosporine induces a mature neuronal noradrenergic phenotype in SH-SY5Y cells through an alpha-, b eta-, and zeta-PKC-independent pathway.