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.