Fig 1.
Structures of a diverse selection of HDACi.
Panobinostat, trichostatin A, and SAHA are pan-isoform inhibitors. Entinostat is a class I selective inhibitor. PCI-34051 is an HDAC8 selective inhibitor.
Fig 2.
Potent HDACi alter the subcellular localization of HDAC1.
MCF-7 cells were treated with indicated concentrations of panobinostat or trichostatin A for 12 hours. A) Western blot analysis of the abundance of HDAC1 in the cytosolic, nuclear soluble, and chromatin bound fractions. B) Densitometry analysis of the abundance of HDAC1 normalized to GAPDH (cytosolic fraction) or to TATA-binding protein (TBP, nuclear soluble and chromatin bound fractions). C) Western blot analysis of the total abundance of class I HDACs and the loading controls TBP, GAPDH, and histone H3 after treatment with indicated concentrations of panobinostat for 12 hours. * Statistically significant difference compared with DMSO control (Student’s t-test, P<0.01). Western blots shown are representative of at least two independent experiments. HDAC1 fold change is presented as the mean of at least two independent experiments ± standard deviation.
Fig 3.
HDACi-induced re-equilibration of HDAC1 is confirmed by confocal microscopy.
MCF-7 cells were treated with indicated concentrations of panobinostat (optical sections A-D, respectively) or trichostatin A (optical sections E-H, respectively) for 12 hours, fixed, permeabilized and optical sections were obtained by laser scanning confocal microscopy. Fluorescence signal for HDAC1 is shown in green (left panels), DAPI staining is shown in blue (middle panels), and merged optical sections are shown in the right panels. Colocalization analysis of HDAC1 fluorescence signal and the DAPI stain signal was performed with JACoP (ImageJ) and shown below. * Statistically significant difference compared with DMSO control (Student’s t-test, P<0.01). Pearson’s Coefficient is presented as the mean of at least two independent experiments ± standard deviation. Optical sections shown are representatives of at least two independent experiments.
Fig 4.
HDAC1 re-equilibration induced by HDACi is subsequent to histone acetylation and is affected by mitogenic stimuli.
A) Western blot analysis of cytosolic fractions of MCF-7 cells treated with indicated concentrations of trichostatin A for 2 hours. B) Densitometry analysis of western blots of chromatin fractions from MCF-7 cells treated with 10 μM trichostatin A for 2 and 12 hours; change in AcH3 was normalized to TBP. C) Western blot analysis of chromatin bound fractions from MCF-7 cells treated with indicated concentrations of HDACi for 12 hours. D) Laser scanning confocal microscopy of MCF-7 cells, grown with 10% serum and treated with DMSO for 12 hours. Fluorescence signal for HDAC1 is shown in green (left panels), DAPI staining is shown in blue (middle panels), and merged optical sections are shown in the right panels. Representative optical section from two independent experiments is shown. Arrows indicate mitotic cells where HDAC1 is dispersed off chromatin, as indicated by DAPI staining. * Statistically significant difference compared with DMSO control (Student’s t-test, P<0.01).
Fig 5.
Increase in cytosolic HDAC1 is irreversible up to 24 hours.
MCF-7 cells were treated with 10 μM panobinostat for 12 hours, the compound was then removed and cells allowed to recover for 24 hours. A) Western blot analysis of the abundance of HDAC1 in the cytosolic, nuclear soluble, and chromatin bound fractions. B) Densitometry analysis of the abundance of HDAC1 normalized to GAPDH (cytosolic fraction) or to TATA-binding protein (TBP, nuclear soluble and chromatin bound fractions). * Statistically significant difference compared with DMSO control (Student’s t-test, P<0.01). Western blots shown are representative of at least two independent experiments. HDAC1 fold change is presented as the mean of at least two independent experiments ± standard deviation.
Fig 6.
Potent HDACi increase the abundance of non-phosphorylated HDAC3.
MCF-7 cells were treated with indicated concentrations of panobinostat, trichostatin A, or SAHA for 12 hours and then biochemically fractionated. A) The abundance of non-phosphorylated HDAC3 was characterized by Western blot analysis in the cytosolic fraction. B) Densitometry analysis of the abundance of non-phosphorylated HDAC3 normalized to GAPDH. * Statistically significant difference compared with DMSO control (Student’s t-test, P<0.01). Western blots shown are representative of at least two independent experiments. HDAC3 fold change is presented as the mean of at least two independent experiments ± standard deviation.
Fig 7.
A model of the mechanism of action of propenamide-based HDACi including re-equilibration of the subcellular distribution and modulation of the post-translational modification of HDACs.
Treatment of with N-hydroxy propenamide-based HDACi increase histone acetylation at 2 hours without affecting the subcellular localization or the phosphorylation status of HDACs. At 12 hours, re-equilibration of subcellular localization as well as a decrease in phosphorylation of HDACs is observed, along with a greater increase in histone acetylation. The induced change in subcellular localization and phosphorylation of HDACs is sustained 24 hours after removal of HDACi. This is in line with the time necessary to induce commitment to cell death with HDACi.