Staurosporine-induced apoptosis presents with unexpected cholinergic effects in a differentiated neuroblastoma cell line |
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Authors: | Guangfeng Li Anne GleinichHelene Lau Martina Zimmermann |
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Affiliation: | Department of Pharmacology, School of Pharmacy, Biocentre N260, Max-von-Laue Str. 9, Goethe University Frankfurt, 60438 Frankfurt am Main, Germany |
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Abstract: | Apoptosis of cholinergic neurons is one of the core hallmarks of Alzheimer’s disease. SH-SY5Y neuroblastoma cells differentiated to the cholinergic phenotype were exposed to 100 nM staurosporine. Over a treatment period of 24 h, the pro- and anti-apoptotic factors, caspase-3 and Bcl-2, as well as LDH release as a measure of cell viability, were assessed in conjunction with the number of apoptotic cells by means of fluorescence-activated cell sorting. Caspase-3 activity and LDH release increased by 30% and 20% over controls, respectively, while Bcl-2 levels rose by 200% over controls. Furthermore, staurosporine treatment resulted in decreased acetylcholinesterase (AChE) enzymatic activity and decreased protein levels of the AChE splice variant tailed AChE (AChE-T). Only a slight increase in levels of readthrough AChE (AChE-R) was observed. Likewise, staurosporine reduced levels and activity of the cholinergic players choline acetyltransferase and high affinity choline uptake. The present study demonstrates that treatment with staurosporine leads to apoptotic events, which, however, are not reflected in the increased AChE activity and the alterations of AChE isoforms expression that are usually seen in apoptotic conditions. The effects of various additional phosphorylation inhibitors on AChE activity suggest that these unexpected cholinergic effects, firstly, are linked to the impact of staurosporine on phosphorylation and, secondly, reveal themselves in a first phase of cellular adaption that precedes neurotoxicity and subsequent cell death. |
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Keywords: | AChE, acetylcholinesterase AChE-T, tailed AChE AChE-R, readthrough AChE AMP-PNP, adenosine 5&prime -(β,γ-imido)triphosphate AV, Annexin V-FITC ChAT, choline acetyltransferase FBS, fetal bovine serum HACU, high affinity choline uptake HC-3, hemicholinium-3 iso-OMPA, tetraisopropyl pyrophosphoramide KHB, Krebs-Henseleit buffer LDH, lactate dehydrogenase MEM, minimum essential medium Eagle mAb, monoclonal antibody MW, molecular weight MWS, molecular weight standard OD, optical density pAb, polyclonal antibody pNA, para-nitro-aniline PBS, phosphate buffered saline PI, propidium iodide PI3K, phosphoinositide 3-kinase PKC, protein kinase C PP2, (4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine RA, retinoic acid SEM, standard error of the mean Stauro, staurosporine |
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