An HDAC3 selective inhibitor triggers apoptosis associated with increased DNA damage and cell cycle defects.

<p>(A) Hut78 cells were treated with DMSO, 10 nM Depsipeptide (Depsi), 10 µM 233, or 10 µM 966 for 24 hr and apoptosis assessed by Annexin V staining and flow cytometry. Cells were also labeled with propidium iodide to assess DNA content. Untreated (UT) and DMSO treated cells were used as controls. Shown is a representative graph from an experiment performed in duplicate that is consistent with other biological replicates. (B) Western blot analysis of γH2aX levels in Hut78 cells treated with DMSO, 10 nM Depsi, 10 µM 233, or 10 µM 966 for 8 hrs. Untreated and DMSO treated cells were used as controls. Samples were run on the same gel and probed on the same membrane. Intervening lanes (represented by a black bar) were removed for side by side comparison of DMSO and Depsipeptide. (C) Cell cycle status was analyzed using BrdU incorporation and propidium iodide to assess DNA content by flow cytometry. Hut78 cells were treated with DMSO, 10 nM Depsipeptide (Depsi), 10 µM 233, or 10 µM 966 for 24 hr and pulsed for an hour and a half with BrdU prior to cell harvest and analysis. Shown are representative flow cytometry plots from an experiment performed in duplicate that is consistent with other biological replicates. (D) Graphical representation of BrdU incorporation from the experiment described in (C). (E) Graphical representation of the percent of S phase cells that did not incorporate BrdU (shown by box in panel (C)). Statistical analysis for both the Annexin V and BrdU experiments was performed using a two-tail T-test and comparing the HDI treated cells to the DMSO treated cells resulting in the following p-values: (A) Depsi: p = 0.0002, 233: p = 0.003, and 966: p = 0.0003. (D) Depsi: p = 0.003, 233: p = 0.01, and 966: p = 0.08. (E) Depsi: p = 0.003, 233: p = 0.003, and 966: p = 0.004.</p

Dual treatment with RGFP966 and CTCL drugs has an additive effect on cell growth.

<p>Growth curves of dual treatment on HH cells or Hut78 cells. Cells were treated once at hour 0 with DMSO, 10 nM Depsipeptide (Depsi), 2 µM 966, or a combination of 2 µM 966 and either Bexarotene, Methotrexate, or ATRA. Untreated cells and DMSO treated cells were used as controls. Cell growth was assessed at 0, 24, 48, and 72 hours after treatment. (A) HH cells (left) or Hut78 cells (right) were treated with 20 µM or 75 µM Bexarotene alone or in combination with 966. (B) Cells were treated with 0.1 µM Methotrexate alone or in combination with 966. DMSO and 1 M Na₂CO₃ served as vehicle controls. (C) Cells were treated with 2 µM ATRA alone or in combination with 966. ATRA was administered at hour 0 and re-dosed at 48 hours after treatment. For (A–C), representative curves are shown from experiments performed in triplicate that are consistent with other biological replicates. Statistical analysis was performed using a two-tail paired T-test and comparing the HDI, CTCL drug, or dual treated cells to DMSO treated cells resulting in the following p values: (A) HH cells (left), Depsi: p = 0.0008, 966: p = 0.003, Bexarotene: p = 0.003, and 966 plus Bexarotene: p = 0.002. For the Hut78 cells (right), Depsi: p = 0.001, 966: p = 0.08, Bexarotene: p = 0.01, and 966 plus Bexarotene: p = 0.009. (B) HH cells (left), Depsi: p = 0.0008, 966: p = 0.003, Methotrexate: p = 0.003, and 966 plus Methotrexate: p = 0.003. For the Hut78 cells (right) Depsi: p = 0.001, 966: p = 0.01, Methotrexate: p = 0.01, and 966 plus Methotrexate: p = 0.004. (C) HH cells (left), Depsi: p = 0.0008, 966: p = 0.003, ATRA: p = 0.002, and 966 plus ATRA: p = 0.0007. For the Hut78 cells (right) Depsi: p = 0.001, 966: p = 0.01, ATRA: p = 0.02, and 966 plus ATRA: p = 0.004.</p

iPOND analysis reveals HDAC3 association with replication forks in Hut78 CTCL cells.

<p>Hut78 cells were pulsed for 15 minutes with EdU followed by either no thymidine chase or a 60 minute thymidine chase. The protein-DNA complexes were then cross-linked, nascent DNA was conjugated to biotin using click chemistry, and then protein-DNA complexes were purified using Streptavidin beads. The eluted proteins were then analyzed using western blot analysis. A no click reaction sample (No Clk) that did not include biotin azide was used as a negative control. 0.1% input samples were included for positive controls of each protein analyzed. PCNA served as a positive control for a replication fork bound protein and H2B served as a loading control and positive control for a chromatin bound protein.</p

HDIs show selective inhibition of HDACs in CTCL cell lines.

<p>(A)Western blot analysis of whole cell lysates from Wild-type (WT) and <i>Hdac3</i>-null livers. Histones H3 and H4 served as loading controls. (B) Upper Panel: Western blot analysis of NIH 3T3 cells following treatment with various HDIs (indicated above each lane). Anti-histone H3 was used as a loading control. Lower panel: Western blot analysis of NIH 3T3 cells treated with either Trichostatin A (TSA) (1 µM), sodium butyrate (NaB) (5 mM), or increasing concentrations of nicotinamide (mM). (C) Western blot analysis of whole cell lysates prepared from cells that were transfected with either non-targeting siRNAs (NT) or siRNAs directed to the indicated Hdacs. (D) Western blot analysis of H3K56ac using whole cell lysates prepared from cells treated with the indicated amounts of RGFP966 for 24 hr. (E & F) Western blot analysis of (E) HH or (F) Hut78 cell lines treated with DMSO, 10 nM Depsipeptide (Depsi), 10 µM 233, 10 µM 136, or 10 µM 966. Cells were treated for 24 hr and then harvested for protein isolation. Samples were run on the same gel and probed on the same membrane. Intervening lanes (represented by a black bar) were removed for side-by-side comparison of DMSO and Depsipeptide. Histones H3 and H4 were used as loading controls.</p

CTCL cell lines are sensitive to pan and selective HDIs.

<p>(A) Growth curves of HDI treated HH cells (left) or Hut78 cells (right). Cells were treated once with DMSO, 10 nM Depsipeptide (Depsi), 10 µM 233, or 10 µM 966 at hour 0. Untreated cells and DMSO treated cells were used as controls. Cell growth was assessed at 0, 24, 48, and 72 hours after treatment. (B) Dose curves of 966 treated HH cells (left) and Hut78 cells (right). The experiment was performed in the same manner as (A) except that the cells treated were treated once with 2 µM, 5 µM, or 10 µM of 966 at hour 0. For both (A) and (B), representative curves are shown from experiments performed in triplicate that are consistent with other biological replicates. Statistical analysis was performed using a two-tail paired T-test and comparing the HDI treated cells to DMSO treated cells resulting in the following p values: (A) HH cells (left), Depsi: p = 0.0008, 233: p = 0.004, and 966: p = 0.006. For the Hut78 cells (right), Depsi: p = 0.002, 233: p = 0.006, and 966: p = 0.006. (B) HH cells (left), Depsi: p = 0.0008, 966 2 µM: p = 0.02, 966 5 µM: p = 0.01, and 966 10 µM: p = 0.006. For the Hut78 cells (right), Depsi: p = 0.002, 966 2 µM: p = 0.03, 966 5 µM: p = 0.01, and 966 10 µM: p = 0.006.</p

HDAC3 selective inhibitors rapidly cause defects in DNA replication.

<p>(A) Western blot analysis of Hut78 cells treated with DMSO or 10 nM Depsipeptide (Depsi) for 4 hrs, or 10 µM 966 for 30 min, 1 hr, 2 hr, and 4 hr. (B, C, and D) DNA fiber labeling analysis was used to assess DNA replication fork progression in Hut78 cells treated with DMSO, 10 nM Depsipeptide (left) or 10 µM 966 (right) for 4 hr (B) or 5 mins (D) prior to labeling with 20 mins of IdU (green) followed by 20 min of CldU (red). Graph of fork velocity (length of fibers divided by 40 min) is shown. (C) Hut78 cells were treated with DMSO, Depsi or 966 immediately after labeling cells with IdU followed by CldU. Graph of fork velocity for either the IdU label or CldU label is shown. Representative fibers are shown. 100 fibers were measured for each sample. Statistical analysis was performed using Mann-Whitney test and standard deviations were calculated. HDI treated cells were compared to DMSO treated cells resulting in the following p-values: (B) Depsi: p<0.0001; 966: p<0.0001. The average velocities for Depsi and 966 were greater than 3 standard deviations of the DMSO average velocity. (C) Depsi IdU (green): p = 0.1, Depsi CldU (red): p = 0.1; 966 IdU (green): p = 0.0011; 966 CldU (red): p = 0.01. The average velocities for IdU and CldU in Depsi treated cells were within 1 and 2 standard deviations respectively of the DMSO average velocity. The average velocities for IdU and CldU in 966 treated cells were within 2 standard deviations of the DMSO average velocity. (D) Depsi: p<0.0001; 966: p<0.0001. The average velocities for Depsi and 966 were greater than 3 standard deviations of the DMSO average velocity.</p