UNIGE document Scientific Article
previous document  unige:120581  next document
add to browser collection
Title

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

Authors
Simões-Pires, Claudia A
Published in Journal of Visualized Experiments. 2017, no. 126, p. e55878
Abstract The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography - mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.
Keywords ChromatographyHigh Pressure LiquidMethodsDepsipeptidesPharmacologyDrug EvaluationPreclinicalHeLa CellsHistone Deacetylase 1AnalysisMetabolismHistone Deacetylase 6Histone Deacetylase InhibitorsHumansHydroxamic AcidsMass Spectrometry
Identifiers
PMID: 28829415
Full text
Article (Published version) (329 Kb) - public document Free access
Other version: https://www.jove.com/video/55878
Structures
Research group Pharmacognosie
Citation
(ISO format)
SIMÕES-PIRES, Claudia A et al. Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS. In: Journal of Visualized Experiments, 2017, n° 126, p. e55878. doi: 10.3791/55878 https://archive-ouverte.unige.ch/unige:120581

149 hits

57 downloads

Update

Deposited on : 2019-07-10

Export document
Format :
Citation style :