ABSTRACT
Protein kinase C (PKC) activation induces neuronal differentiation of SH-SY5Y neuroblastoma cells. This study examines the role of PKCbeta isoforms in this process. The PKCbeta-specific inhibitor LY379196 had no effect on 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced neurite outgrowth from SH-SY5Y neuroblastoma cells. On the other hand, PKCbeta inhibition suppressed the TPA-stimulated increase in neuropeptide Y mRNA, activation of neuropeptide Y gene promoter elements, and phosphorylation of Erk1/2. The TPA-induced increase in neuropeptide Y expression was also inhibited by the MEK inhibitor PD98059. These data indicate that activation of a PKCbeta isoform, through a pathway involving Erk1/2, leads to increased expression of neuronal differentiation genes in neuroblastoma cells.
Subject(s)
Cell Differentiation , Isoenzymes/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinases/metabolism , Neurons/physiology , Protein Kinase C/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Culture Media, Serum-Free , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , GAP-43 Protein/metabolism , Gene Expression Regulation/physiology , Genes, Reporter , Isoenzymes/antagonists & inhibitors , Mesylates/pharmacology , Mitogen-Activated Protein Kinase 3 , Neurites/metabolism , Neuroblastoma , Neurons/cytology , Neuropeptide Y/genetics , Neuropeptide Y/metabolism , Phosphorylation , Promoter Regions, Genetic , Protein Kinase C/antagonists & inhibitors , Protein Kinase C beta , Pyrroles/pharmacology , Tumor Cells, CulturedABSTRACT
To investigate a putative involvement of protein kinase C (PKC) isoforms in supporting neuroblastoma cell proliferation, SK-N-BE(2) neuroblastoma cells were transfected with expression vectors coding for the C2 and V5 regions from different PKC isoforms. These structures have been suggested to inhibit the activity of their corresponding PKC isoform. The PKC fragments were fused to enhanced green fluorescent protein to facilitate the detection of transfected cells. Expression of the C2 domain from a classical PKC isoform (PKCalpha), but not of C2 domains from novel PKCdelta or PKCepsilon, suppressed the number of neuroblastoma cells positive for cyclin A and bromodeoxyuridine incorporation. This indicates a role for a classical isoform in regulating proliferation of these cells. Among the V5 fragments from PKCalpha, PKCbetaI, and PKCbetaII, the PKCbetaI V5 had the most suppressive effect on proliferation markers, and this fragment also displaced PKCbetaI from the nucleus. Furthermore, a PKCbeta-specific inhibitor, LY379196, suppressed the phorbol ester- and serum-supported growth of neuroblastoma cells. There was a marked enhancement by LY379196 of the growth-suppressive and/or cytotoxic effects of paclitaxel and vincristine. These results indicate that PKCbetaI has a positive effect on the growth and proliferation of neuroblastoma cells and demonstrate that inhibition of PKCbeta may be used to enhance the effect of microtubule-interacting anticancer agents on neuroblastoma cells.
Subject(s)
Isoenzymes/physiology , Neuroblastoma/metabolism , Neuroblastoma/pathology , Protein Kinase C/physiology , Antineoplastic Agents, Phytogenic/pharmacology , Blotting, Western , Bromodeoxyuridine/metabolism , Cell Differentiation , Cell Division , Cell Survival , Cyclin A/metabolism , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Isoenzymes/chemistry , Microscopy, Fluorescence , Models, Biological , Paclitaxel/pharmacology , Plasmids/metabolism , Protein Isoforms , Protein Kinase C/chemistry , Protein Kinase C beta , Protein Structure, Tertiary , Tumor Cells, Cultured , Vincristine/pharmacologyABSTRACT
To investigate the role of protein kinase C (PKC) isoforms in regulation of neurite outgrowth, PKCalpha, betaII, delta, and epsilon fused to enhanced green fluorescent protein (EGFP) were transiently overexpressed in neuroblastoma cells. Overexpression of PKCepsilon-EGFP induced cell processes whereas the other isoforms did not. The effect of PKCepsilon-EGFP was not suppressed by the PKC inhibitor GF109203X. Instead, process formation was more pronounced when the regulatory domain was introduced. Overexpression of various fragments from PKCepsilon regulatory domain revealed that a region encompassing the pseudosubstrate, the two C1 domains, and parts of the V3 region were necessary and sufficient for induction of processes. By deleting the second C1 domain from this construct, a dominant-negative protein was generated which suppressed processes induced by full-length PKCepsilon and neurites induced during retinoic acid- and growth factor-induced differentiation. As with neurites in differentiated neuroblastoma cells, processes induced by the PKCepsilon- PSC1V3 protein contained alpha-tubulin, neurofilament-160, and F-actin, but the PKCepsilon-PSC1V3-induced processes lacked the synaptic markers synaptophysin and neuropeptide Y. These data suggest that PKCepsilon, through its regulatory domain, can induce immature neurite-like processes via a mechanism that appears to be of importance for neurite outgrowth during neuronal differentiation.
Subject(s)
Isoenzymes/metabolism , Neurites/physiology , Protein Kinase C/metabolism , Actins/metabolism , Animals , Binding Sites , COS Cells , Catalytic Domain , Cell Division , Green Fluorescent Proteins , Humans , Intracellular Fluid , Isoenzymes/genetics , Luminescent Proteins/metabolism , Neuroblastoma , Protein Kinase C/genetics , Protein Kinase C-epsilon , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Tumor Cells, CulturedABSTRACT
To elucidate the possibility of utilizing protein kinase C (PKC) isoforms as target genes in neuroblastoma therapy, 5 neuroblastoma cell lines and neuroblastoma tumor specimens were examined for PKC isoform expression pattern and the cell lines were analyzed for sensitivity to PKC inhibition. All cell lines [IMR-32, LAN-2, LAN-5, SH-SY5Y and SK-N-BE(2)] expressed alpha, betaII, delta and epsilon isoforms of PKC, while no PKCeta or theta protein was detected in any cell line. PKCgamma was found only in LAN-2 cells. PKCalpha, betaII and delta were detected in 5 neuroblastoma tumors and PKCepsilon in 4 out of 5 tumors. Exposure to the PKC inhibitors GF109203X, Gö 6976 or Gö 6983 caused a decrease whereas activation of PKC with 12-O-tetradecanoyl phorbol 13-acetate caused an increase in the number of neuroblastoma cells. The effect of Gö 6976 was due to both inhibited proliferation and to increased apoptosis. While GF109203X suppressed neurite outgrowth induced by a growth factor combination, Gö 6976 potentiated neurite outgrowth. Our data suggest a role for classical PKC isoforms in neuroblastoma growth and survival and for novel isoforms in neurite outgrowth.
