phorbol-12-13-didecanoate and Neuroblastoma

In this study, we investigated the vasoactive intestinal polypeptide (VIP)-stimulated cAMP production and its interaction with protein kinase C activation and elevation of intracellular Ca2+ in N1E-115 neuroblastoma cells. VIP treatment caused a 55-fold increase in cAMP accumulation. Addition of 4 beta-phorbol 12-myristate 13-acetate reduced VIP- but not forskolin-stimulated cAMP response. In comparison, ionomycin potentiated both VIP- and forskolin-induced cAMP accumulation. Our results indicate that VIP stimulates cAMP accumulation in N1E-115 cells, and that although activation of protein kinase C inhibits the VIP-stimulated cAMP response, elevation of intracellular Ca2+ potentiates this signaling pathway.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 1-Methyl-3-isobutylxanthine; Animals; Calcium; Carcinogens; Colforsin; Cyclic AMP; Dose-Response Relationship, Drug; Drug Interactions; Drug Synergism; Enzyme Activation; Ionomycin; Isoquinolines; Kinetics; Mice; Neuroblastoma; Phorbol Esters; Piperazines; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

When established in culture, human neuroblastoma cell lines typically are comprised of heterogeneous cellular subpopulations, including neuroblastic (N-type), substrate-adherent (S-type), and intermediate (I-type) cells that can be distinguished by their characteristic morphologies and expression of differentiation-associated antigens. Here we examined the relative levels of the Bcl-2 oncoprotein in 15 clones derived from four different neuroblastoma cell lines. Among six clones isolated from the SK-N-SH line, levels of p26-Bcl-2 correlated with morphology and differentiation markers with the hierarchy of bcl-2 expression being: N-type cells > N/I-type > I-type > S-type. Furthermore, stimulation of one of the N-type clones, SH-SY5Y, with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, induced differentiation toward a more neuronal-like phenotype and resulted in a 5- to 10-fold elevation in the relative levels of Bcl-2 protein. High relative amounts of p26-Bcl-2 protein were also found in an N-type clone derived from the SMS-KCN line. In two N-type clones derived from the LA-N-1 line, however, levels of Bcl-2 protein were only moderately elevated, and in one N-type clone from the SK-N-BE(2) line the levels of Bcl-2 protein were low. Thus, high relative levels of Bcl-2 oncoprotein are not a universal feature of N-type cells (three of six clones tested). In contrast, all 5 of the S-type clones evaluated contained relatively low levels of Bcl-2 protein, suggesting that these cells (which may represent embryonic precursors of Schwann, glial, and melanocytic cells) do not typically express the bcl-2 gene at high levels. Consistent with this inverse correlation between Bcl-2 protein levels and S-type characteristics, stimulation of an I-type clone derived from the SK-N-BE(2) line with 5-bromodeoxyuridine was accompanied by an accumulation of S-type cells in these cultures, decreased Bcl-2 protein, diminutions in the neuronal markers neurofilament-M and neuron-specific enolase, and an increase in the relative levels of the S-type marker proteins vimentin and beta-2-microglobulin. Conversely, stimulation of this I-type clone with retinoic acid resulted in an accumulation of N-type cells (which are thought to represent embryonic precursors of sympathetic neurons), decreased vimentin and beta-2-microglobulin, increased neurofilament-M, and a marked elevation in p26-Bcl-2.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Antineoplastic Agents; Carcinogens; Cell Differentiation; Cell Division; Cell Line; Cell Survival; Clone Cells; Drug Resistance; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Neuroblastoma; Oncogenes; Phorbol Esters; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tetradecanoylphorbol Acetate; Time Factors; Transfection; Tretinoin; Tumor Cells, Cultured

Topics: Animals; Carbachol; Cell Line; Cyclic GMP; Inositol Phosphates; Mice; Neuroblastoma; Phorbol Esters; Phorbols; Protein Kinase C; Receptors, Muscarinic; Sugar Phosphates; Tetradecanoylphorbol Acetate

Many recent reports have indicated that the effect of the phorbol ester tumor promoters is mediated through the Ca2+/phospholipid dependent protein kinase C. We have investigated the effect of two biologically active phorbol esters, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) and 4 beta-phorbol 12 beta,13 alpha-didecanoate (beta PDD) on muscarinic agonist binding and receptor-stimulated phosphoinositide breakdown in cultured human neuroblastoma (SH-SY5Y) cells. Preincubation of these cells with phorbol esters significantly reduced the carbachol-stimulated breakdown of inositol phospholipids and caused a decrease of agonist affinity for [3H](-)methyl quinuclidinyl benzilate ([3H](-)MQNB) binding without affecting the affinity of antagonist to the muscarinic receptor. The nontumor promoting 4 alpha-phorbol 12 beta,12 alpha-didecanoate (alpha PDD) was ineffective in our studies. These results suggest that the activation of protein kinase C may play an important role in regulating the muscarinic receptor system.

Topics: Atropine; Cell Line; Humans; Neuroblastoma; Phorbol Esters; Phosphatidylinositols; Protein Kinase C; Receptors, Muscarinic; Tetradecanoylphorbol Acetate