Distinct Mechanisms of Differentiation of SH‐SY5Y Neuroblastoma Cells by Protein Kinase C Activators and Inhibitors

Abstract

Certain biological actions of phorbol esters cannot be duplicated by diacylglycerol (DAG). Thus, the human neuroblastoma cell line SH-SY5Y differentiates when exposed to 12-tetradecanoyl-13-acetyl-beta-phorbol (TPA) and protein kinase C (PKC) inhibitors, but not when exposed to DAG. To investigate the specific features of the phorbol diester molecule that might be responsible for these effects, we examined the extension of neurites, expression of neuron-specific enolase, and appearance and localization of phosphorylated high molecular weight neurofilament subunits (NF-H). TPA, 12-deoxy-13-tetradecanoyl-beta-phorbol, and staurosporine, but not DAG or 4-O-methyl-TPA, caused neurite outgrowth. Neuron-specific enolase was expressed in cells treated with TPA and 12-deoxy-13-tetradecanoyl-beta-phorbol but not with DAG, staurosporine, or 4-O-methyl-TPA. NF-H increased in the perikarya of cells treated with DAG and 4-O-methyl-TPA, in processes and to varying degrees in perikarya of TPA- and 12-deoxy-13-tetradecanoyl-beta-phorbol-treated cells, but much more in the processes than in the perikarya of staurosporine-differentiated cells. These findings and additional differences between the differentiation induced by TPA (a PKC activator) and staurosporine (a PKC inhibitor), including distinct morphology of the cell body and processes and time of appearance of the morphological phenotype, suggest that activators and inhibitors of PKC induce differentiation of SH-SY5Y cells by different mechanisms, and that the five-membered/seven-membered terpene ring region present in TPA must be intact for the induction of morphological differentiation.

Intracellular delivery of protein kinase C‐α or ε isoform‐specific antibodies promotes acquisition of a morphologically differentiated phenotype in neuroblastoma cells

“…Incubations were continued as indicated. At the end of the incubation cells were fixed and examined by phase-contrast microscopy [23]. When antibodies were pre-absorbed with antigen peptides, the pcptides in three-fold excess were mixed whh the antibodies at least 5 min before USC.…”

“…When antibodies were pre-absorbed with antigen peptides, the pcptides in three-fold excess were mixed whh the antibodies at least 5 min before USC. For visualization of antibodies in the treated cultures, cells were immunostaincd with a secondary biotinylated goat anti-rabbit antibody, using the ABC system as described [23].…”

“…Differentiation of SM-SYTY cells induced by TPA is almost complete [ 151, involving down-regulation of the expression of c-n?yc and increase of the expression of c-fos [20], followed by appearance of voltage-dependent Ca? '-channels [21], neuron-specific enolase [18,21], norepinephrine [22], and neurites [IS], containing stable cytoskeleton [23].…”

Stromal heparan sulfate differentiates neuroblasts to suppress neuroblastoma growth

“…Since surface TβRIII expression promotes neuroblast differentiation via extracellular glycosaminoglycan modifications (12), we investigated whether soluble HSPGs (sHSPGs) could have similar effects. We used well-established models of neuroblast differentiation: neurite outgrowth in 5Y and BE2 cells (36,37) and expression of neuron-specific proteins, including neurofilament 160 kDa in 5Y, BE2, and SK-N-AS cells (10,(38)(39)(40), neuron-specific enolase or tyrosine hydroxylase in 5Y cells (10,41), and β3-tubulin in BE2 and SK-N-AS cells (42). Recombinant sTβRIII, sGPC1, sGPC3, sSDC3, and sSDC4 all enhanced neurite outgrowth in 5Y neuroblastoma cells (Figure 2, A and B, and Supplemental Figure 3, A-C).…”

Rit promotes MEK-independent neurite branching in human neuroblastoma cells

“…These cells, originally subcloned from a metastatic neuroblastoma, represent a homogenous culture of sympathetic noradrenergic neurons (Ross et al, 1983) and emulate the behavior and biology of primary neurons (Ross et al, 1983;Påhlman et al, 1990;Leli et al, 1992;Påhlman et al, 1992;Choi et al, 1994;Leventhal and Feldman, 1995;Smith et al, 1995;Hynds et al, 1997;Hynds and Snow, 1999;Zeidman et al, 1999;Encinas et al, 2000;Hynds and Snow, 2001;Snow et al, 2001). Adenovirus-mediated expression of CA-Rit induced extensive neurite outgrowth and, importantly, branching in SH-SY5Y cells cultured on endogenous extracellular matrix or purified laminin-1.…”