title: Detection and quantification of farnesol-induced apoptosis in difficult primary cell cultures by TaqMan protein assay.
abstract: Apoptosis can be detected reliably by assaying for cleaved caspase-3, for which active caspase-3 antibodies are used in several methods, such as immunocytochemistry, enzyme-linked immunosorbent assay, and western blot. In this study, we used TaqMan protein assay (TPA), a novel method for protein detection and quantification that detects proteins by amplification of substitute DNA templates. TPA uses antibodies and proximity ligation for quantitative real-time PCR. Meningiomas are primarily benign intracranial tumors. Primary cell cultures of meningiomas are often unsuitable for sensitive protein detection methods. We optimized a TPA to detect active caspase-3 and evaluated its ability to detect farnesol-induced apoptosis in primary meningioma cells. The specificity and sensitivity of the inactive and active caspase-3 assay were determined using recombinant caspase-3. Apoptosis was induced in meningiomas in the presence of 0.2 μM farnesol as shown by immunocytochemistry of single-stranded DNA. Also, viability decreased by over 90 % after treatment with 1.2 μM farnesol for 24 h. The TPA detected a significant increase in active caspase-3 after treatment with 2 and 4 μM farnesol for 2 h, which could not be detected using standard methods such as western blot and immunofluorescence. In addition, TPA determined that meningiomas show disparate sensitivities to low concentrations of farnesol. Caspase-3 expression fell significantly in cells that were treated with 0.25 μM farnesol for 2 h. Further, by TPA, active caspase-3 peaked after 2 h and declined with longer incubation times. This study demonstrates that cleaved caspase-3 is detected and quantified reliably in meningiomas by TPA.
authors: Department of Neurosurgery, University of Tuebingen, Hoppe-Seyler-Str. 3, 72076 Tuebingen, Germany. christina.pfister@med.uni-tuebingen.de
authors: Christina
authors: C
authors: Pfister
authors: Heike
authors: H
authors: Pfrommer
authors: Marcos S
authors: MS
authors: Tatagiba
authors: Florian
authors: F
authors: Roser
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000235:genetics; D005204:Farnesol / Q000494:pharmacology*; D005260:Female; D006801:Humans; D008297:Male; D008579:Meningioma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
chemical_list: D004277:DNA, Single-Stranded; D005204:Farnesol; D053148:Caspase 3