Expression of Protein Kinase C Isoforms in Euxanthone-Induced Differentiation of Neuroblastoma Cells

Abstract

Euxanthone, a potent neuritogenic compound isolated from the roots of the medicinal herb Polygala caudata, has recently been shown to induce the differentiation of murine neuroblastoma Neuro 2A (BU-1) cells. In this study, the role of protein kinase C (PKC) and the expression of various PKC isoforms in euxanthone-treated BU-1 cells were examined. mRNA phenotyping using the reverse-transcription polymerase chain reaction (RT-PCR) showed that BU-1 cells express six different PKC isoforms, namely PKC-α, -β, -δ, -ε, -λ, and -ζ. Differential regulation and expression of PKC isoforms was observed in BU-1 cells treated with 100 μM euxanthone. PKC-α, -β, -δ, -λ and -ζ were all up-regulated, with 1.7- to 9.5-fold increase, at around 30 to 60 minutes after euxanthone treatment. The expression level of PKC-ε remained relatively constant during the treatment. PKC-γ, -η, and -Θ were not detected in both untreated and euxanthone-treated BU-1 cells. Staurosporine, a broad spectrum PKC inhibitor, was found to inhibit both spontaneous and euxanthone-induced neuritogenesis in BU-1 cells. A significant reduction of the euxanthone-induced neuritogenic effect was also observed when the PKC isoform-specific inhibitor Go6976 was included in the culture. These results suggest that the euxanthone-induced differentiation of the neuroblastoma BU-1 cells may be mediated through the differential expression of PKC-α, -β, -δ, -λ and -ζ isoforms.

Key words

Protein kinase C isoforms - euxanthone - differentiation - neuroblastoma cells

References

• 1 Mellor H, Parker P J. The extended protein kinase C superfamily.  Biochemical Journal. 1998;  332 281-92
• 2 Mohan D RK, Nagarathna R, Krishna M, Jagtap J C, Shastry P. Differential responses of staurosporine on protein kinase C activity and proliferation in two murine neuroblastoma cell lines.  Cancer Letters. 1999;  139 137-43
• 3 Zeidman R, Pettersson L, Sailaja R, Truedsson E, Fagerstrom S. Novel and classical protein kinase C isoforms have different functions in proliferation, survival and differentiation of neuroblastoma cells.  Internal Journal of Cancer. 1999;  81 494-501
• 4 Fagerstrom S, Pahlman S, Gestblom C, Nanberg E. Protein kinase C-epsilon is implicated in neurite outgrowth in differentiating human neuroblastoma cells.  Cell Growth and Differentiation. 1996;  7 775-85
• 5 Li W K, Chan H W, Leung H W, Yeung H W, Xiao P G. Xanthones and flavonoids of Polygala caudata .  Pharmacy and Pharmacology Communications. 1998;  4 415-7
• 6 Mak N K, Li W K, Zhang M, Wong R NS, Tai L S, Yung K KL, Leung H W. Effects of euxanthone on neuronal differentiation.  Life Sciences. 2000;  66 347-54
• 7 Mak N K, Szeto Y Y, Fung M C, Leung K N, Kwan S K. Effects of midazolam on the differentiation of murine myeoloid leukemia cells.  Chemotherapy. 1997;  43 272-81
• 8 Freire-Moar J, Cherwinski H, Hwang F, Ransom J, Webb D. Expression of protein kinase C isoenzymes in thymocyte subpopulations and their differential regulation.  Journal of Immunology. 1991;  147 405-9
• 9 Krizbai I, Szabo G, Deli M, Maderspach K, Lehel C, Olah Z, Wolff J R, Joo F. Expression of protein kinase C family members in the cerebral endothelial cells.  Journal of Neurochemistry. 1995;  65 459-62
• 10 Gangeswaran R, Jones K T. Unique protein kinase C profile in mouse oocytes: lack of calcium-dependent conventional isoforms suggested by rtPCR and Western blotting.  FEBS Letters. 1997;  412 309-12
• 11 Shibata M, Hariya T, Hatao M, Ashikaga T, Ichikawa H. Quantitative polymerase chain reaction using an external control mRNA for determination of gene expression in a heterogeneous cell population.  Toxicology Sciences. 1999;  49 290-6
• 12 Ekinci F J, Shea T B. Selective activation by bryostatin-1 demonstrates unique roles for PKC epsilon in neurite extension and tau phosphorylation.  International Journal of Developmental Neuroscience. 1997;  15 867-74
• 13 Ghigo D, Priotto C, Migliorino D, Geromin D, Franchino C, Todde R, Costamagna C, Pescarmona G, Bosia A. Retinoic acid-induced differentiation in a human neuroblastoma cell line is associated with an increase in nitric oxide synthesis.  Journal of Cell Physiology. 1998;  174 99-106
• 14 O'Driscoll K R, Teng K K, Fabbro D, Greene L A, Weinstein I B. Selective translocation of protein kinase C-delta in PC12 cells during nerve growth factor induced neuritogenesis.  Molecular Biology of Cell. 1995;  6 449-58
• 15 Zeidman R, Lofgren B, Pahlman S, Larsson C. PKC epsilon, via its regulatory domain and independently of its catalytic domain, induces neurite-like processes in neuroblastoma cells.  Journal of Cell Biology. 1999;  145 713-26
• 16 Owen P J, Johnson G D, Lord J M. Protein kinase C delta associates with vimentin intermediate filaments in differentiated HL60 cells.  Experimental Cell Research. 1996;  225 366-73
• 17 Wang Q J, Acs P, Goodnight J, Giese T, Blumberg P M, Mischak H, Mushinski J F. The catalytic domain of protein kinase C delta in reciprocal delta and epsilon chimeras mediates phorbol ester-induced macrophage differentiation of mouse promyelocytes.  Journal of Biological Chemistry. 1997;  272 76-82
• 18 Wooten M W. Function for NF-kB in neuronal survival: regulation by atypical protein kinase C.  Journal of Neuroscience Research. 1999;  58 607-11
• 19 Wooten M W, Seibenhener M L, Zhou G, Vandenplas M L, Tan T H. Overexpression of atypical PKC in PC12 cells enhances NGF-responsiveness and survival through an NF-kappaB dependent pathway.  Cell Death and Differentiation. 1999;  6 753-64
• 20 Bareggi R, Bratina F, Grill V, Narducci P, Martelli A M. Atypical isoenzymes of PKC-iota, -lambda, -mu: relative distribution in mouse foetal and neonatal organs.  Italian Journal of Anatomy and Embryology. 1998;  103 127-43
• 21 Martiny-Baron G, Kazanietz M G, Mischak H, Blumberg P M, Kochs G, Hug H, Marme D, Schachtele C. Selective inhibition of protein kinase C isozymes by the indolocarbazole Go6976.  Journal of Biological Chemistry. 1993;  268 9194-7