class i hdac imaging using [3h]ci-994 autoradiography

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Class I HDAC imaging using [3H]CI-994 autoradiographyYajie Wanga, Yan-Ling Zhangb, Krista Hennigc, Jennifer P. Galeb, Yijia Honga, Anna Chaa,Misha Rileya, Florence Wagnerb, Stephen J. Haggartyc, Edward Holsonb & Jacob Hookera

a Athinoula A. Martinos Center; Department of Radiology; Massachusetts General Hospital;Harvard Medical School; Charlestown, MA USAb Stanley Center for Psychiatric Research; Broad Institute of Harvard and MIT; Cambridge,MA USAc Center for Human Genetic Research; Simches Research Center; CPZN-5242, Departments ofNeurology & Psychiatry; Harvard Medical School; Boston, MA USAPublished online: 11 Jun 2013.

To cite this article: Yajie Wang, Yan-Ling Zhang, Krista Hennig, Jennifer P. Gale, Yijia Hong, Anna Cha, Misha Riley, FlorenceWagner, Stephen J. Haggarty, Edward Holson & Jacob Hooker (2013) Class I HDAC imaging using [3H]CI-994 autoradiography,Epigenetics, 8:7, 756-764, DOI: 10.4161/epi.25202

To link to this article: http://dx.doi.org/10.4161/epi.25202

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Epigenetics 8:7, 756–764; July 2013; © 2013 Landes Bioscience

RESEARCH PAPER

756 Epigenetics Volume 8 Issue 7

*Correspondence to: Jacob Hooker; Email: [email protected]: 03/21/13; Revised: 05/10/13; Accepted: 05/29/13http://dx.doi.org/10.4161/epi.25202

Introduction

Several enzymes control the structure of chromatin and the accessibility of its associated DNA for transcription.1 Of these, two families of enzymes modify the charge state of histone tails through acetylation or deacetylation of lysine residues. Although these enzymes can be promiscuous and are known to react with many non-histone proteins,2 many of their primary biological functions have been ascribed to chromatin remodeling. Histone acetyltransferases (HATs) function by transferring the acetyl group of acetyl coenzyme A to the ε-amino group of certain lysines, which results in neutralization of the normally cationic amino acid. This in turn facilitates chromatin relaxation and can provide DNA access to the transcriptional machinery, as well as lead to the recruitment of acetyl-lysine binding proteins. HAT activity is opposed by histone deacetylases (HDACs), which trigger chromatin condensation and transcriptional repression.3 Dysfunctional regulation of histone acetylation has been impli-cated in the pathophysiology of several diseases including can-cer, heart failure, inflammation and neurodegeneration.4 In some

[3H]CI-994, a radioactive isotopologue of the benzamide CI-994, a class I histone deacetylase inhibitor (HDACi), was evaluated as an autoradiography probe for ex vivo labeling and localizing of class I HDAC (isoforms 1–3) in the rodent brain. After protocol optimization, up to 80% of total binding was attributed to specific binding. Notably, like other benzamide HDACi, [3H]CI-994 exhibits slow binding kinetics when measured in vitro with isolated enzymes and ex vivo when used for autoradiographic mapping of HDAC1–3 density. The regional distribution and density of HDAC1–3 was determined through a series of saturation and kinetics experiments. The binding properties of [3H]CI-994 to HDAC1–3 were characterized and the data were used to determine the regional Bmax of the target proteins. Kd values, determined from slice autoradiography, were between 9.17 and 15.6 nM. The HDAC1–3 density (Bmax), averaged over whole brain sections, was of 12.9 picomol·mg−1 protein. The highest HDAC1–3 density was found in the cerebellum, followed by hippocampus and cortex. Moderate to low receptor density was found in striatum, hypothalamus and thalamus. These data were correlated with semi-quantitative measures of each HDAC isoform using western blot analysis and it was determined that autoradiographic images most likely represent the sum of HDAC1, HDAC2, and HDAC3 protein density. In competition experiments, [3H]CI-994 binding can be dose-dependently blocked with other HDAC inhibitors, including suberoylanilide hydroxamic acid (SAHA). In summary, we have developed the first known autoradiography tool for imaging class I HDAC enzymes. Although validated in the CNS, [3H]CI-994 will be applicable and beneficial to other target tissues and can be used to evaluate HDAC inhibition in tissues for novel therapies being developed. [3H]CI-994 is now an enabling imaging tool to study the relationship between diseases and epigenetic regulation.

Class I HDAC imaging using [3H]CI-994 autoradiography

Yajie Wang,1 Yan-Ling Zhang,2 Krista Hennig,3 Jennifer P. Gale,2 Yijia Hong,1 Anna Cha,1 Misha Riley,1 Florence Wagner,2 Stephen J. Haggarty,3 Edward Holson2 and Jacob Hooker1,*

1Athinoula A. Martinos Center; Department of Radiology; Massachusetts General Hospital; Harvard Medical School; Charlestown, MA USA; 2Stanley Center for Psychiatric Research; Broad Institute of Harvard and MIT; Cambridge, MA USA; 3Center for Human Genetic Research; Simches Research Center;

CPZN-5242, Departments of Neurology & Psychiatry; Harvard Medical School; Boston, MA USA

Keywords: autradiography, HDAC, CI-994, class I, inhibitor, benzamide, density

cases, the acetylation status can be “rescued” through chemical inhibition of certain HDACs, a strategy that has been success-ful in cancer therapy.5-7 The utility of HDACi in treating central nervous system (CNS) disorders is less clear;8 however, abnormal expression levels of class-I HDACs have been correlated with sev-eral CNS disorders, including Huntington disease,9 amyotrophic lateral sclerosis (ALS),10-12 psychiatric disorders,13 and Alzheimer disease.14

Class I HDACs, consisting of HDAC isoforms 1, 2, 3 and 8, appear to have critical functions in brain development and CNS maintenance.15 For example, HDAC2 silences progenitor tran-scripts during neuronal differentiation of adult generated neu-rons16,17 and negatively regulates memory formation and synaptic plasticity. Class I HDAC inhibition can protect against epigene-tic dysfunctions, such as contextual memory defects.18,19 HDAC2 and 3 enzymes have been implicated in neurotoxicity and HDAC inhibitors have potential as neuroprotective drugs.20 HDAC1 and HDAC2 are indispensable for diverse aspects of neuronal development and control the progression of neural precursors to neurons during brain development.21

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www.landesbioscience.com Epigenetics 757

RESEARCH PAPER RESEARCH PAPER

or isoform selective inhibitors, and to moni-tor enzyme density changes as a function of natural variable (e.g., aging and sex) or disease and treatment. Herein, we describe the development and evaluation of an auto-radiography probe, [3H]CI-994, that can be used to measure HDAC1–3 density and dis-tribution in the rodent brain. Furthermore, we demonstrate that [3H]CI-994 can be used in competition experiments to evaluate and explore the affinity and localization of binding for other HDAC inhibitors.

Results and Discussion

Ligand selection and in vitro properties. Autoradiography methods provide a means to determine quantitative maps of protein

density provided that a ligand can be developed, which is both selective and specific (saturable). Ideally, a radioligand should have the following desirable properties: high affinity for its target protein(s), high specific radioactivity (which is obviously isotope-dependent), high specific to non-specific binding ratio (deter-mined through saturation), and high selectivity for its target(s). Our studies began by analyzing the structures and physiochemi-cal properties of ligands known to bind HDAC enzymes with high affinity. We were particularly drawn to molecules known to be selective for class I HDACs due to the role of this class in brain disorders (but generally applicable in other tissues as well). We identified CI-994 as a tractable molecule for tritium label-ing after first carefully analyzing its binding properties in vitro. This began with an analysis that we believed would be most pre-dictive of non-specific binding, namely brain homogenate bind-ing. CI-994 was compared with other benzamide-based HDAC inhibitors in this assay and found to be unique (Fig. 1). Only 12.9% of CI-994 was bound to mouse plasma protein when allowed to equilibrate with an equal volume of buffer; 14% was bound to mouse homogenate. Moreover, we note that the ligand has similarly good dissociation from human plasma proteins (23.7% bound at equilibrium). These data suggested to us that we would be able to wash away non-specifically bound CI-994 in autoradiography assays by using a series of washing steps.

The affinity of CI-994 was determined against the HDAC isoforms 1–3 using a kinetic assay. This provided us with not only the relevant dissociation constant (measured as K

i to be

55, 250, 24 nM, respectively), but also informative information about autoradiography protocol design. These K

i values were

similar to those calculated using a trypsin coupled assay with an H4K12Ac peptide substrate (50, 190, 500 nM, respectively, for HDAC1, 2, 3, with the greatest difference for HDAC3 (~20×). Through these binding kinetics experiments we determined that the association of CI-994 was likely slow (inhibition was slow and time-dependent) and, thus, autoradiography incubation would need to be significantly longer than is common. The long dissociation half-life was, accordingly, very long (58–250 min at room temperature) and provided us with an opportunity for

Although class I HDACs are known to be expressed ubiqui-tously in the CNS and peripheral organs, information about dis-tribution and density is limited to the relative abundance of its associated mRNA and relative HDAC protein density, as deter-mined by techniques such as western blot and immunoprecipita-tion.22-26 Absolute protein densities and distributions of HDAC have not been determined, although these data would enlighten our understanding of HDAC function in both healthy and dis-eased tissue. Moreover, these data would provide a foundation for the development of in vivo imaging agents that could be used to characterize HDAC density, distribution, and ultimately function in humans.27,28 As a stepping stone to these goals, we are develop-ing autoradiography probes that can be used for ex vivo tissue analyses. Development of an autoradiography probe for HDAC has certain advantages and provides complementary information when compared with other ‘visualization techniques’ for HDAC. For example, binding is stoichiometric and quantitative (in an absolute molarity sense); binding is displaceable and can be used to probe small-molecule interactions with HDAC; binding (for the ligand described herein) occurs in the active site and thus the images have direct relevance to the availability of the enzymes for catalysis, which can be important given that perhaps not all com-plex forms of HDAC can ‘receive’ a ligand or perform catalysis. Herein, we describe the development and validation of an auto-radiography ligand, [3H]CI-994, that can be used to characterize class I HDAC enzymes (specifically the sum of HDAC isoforms 1–3) ex vivo.

CI-994, (acetylamino)-N-(2-aminophenyl) benzamide, has demonstrated antitumor activity in vitro and in vivo against a broad spectrum of murine and human tumor models.29-35 CI-994 has also been pursued as a treatment for neurodegenerative dis-eases and for diseases outside of the brain.36,37 For example, it was recently reported that CI-994 inhibition of HDACs may have potential as for the treatment of memory/cognition and anxi-ety disorders (Tsai, et al. submitted) CI-994 is a class I HDACs inhibitor, selective for HDAC1, 2 and 3 with varying potency.39 Thus, a tritium-labeled version could provide a map of HDAC1–3 and be used to associate this class with disease, to develop class

Figure 1. Structure and properties of HDAC inhibitor CI-994. (A) CI-994 is a benzamide inhibitor with MW 269 Da, CLogP of 0.74, and tPSA of 84. (B) CI-994 inhibits recombinant class I HDAC enzymes at nanomolar concentrations with excellent selectivity over other HDAC isoforms. Of note, CI-994 has low plasma protein and brain tissue binding at equilibrium against buffer. (M = mouse; H = human).

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Bmax

of 13.6 picomol·mg−1 protein and Kd 9.6 nM. These data

indicate that the affinity of CI-994 for HDAC in tissue is actually higher than for the isolated recombinant enzymes in vitro and may suggest that HDAC in association with its known binding partners (e.g., in the CoREST complex) may exhibit different binding affinities (and perhaps different selectivity). Moreover, these data support that HDAC class I enzymes are of sufficient concentration in the brain to warrant non-invasive PET imag-ing (ultimately, in humans). PET imaging of HDAC enzymes is a current area of research; however, to date, the B

max was not

known.Validation of imaging data. In order to provide proof that the

autoradiography maps indeed relate to the HDAC1–3 density, we measured the HDAC1, HDAC2, and HDAC3 density in rodent brain regions using ‘semi-quantitative’ western blots. Brains from five different mice were dissected into nine major regions by the method of Glowinski and Iverson (1966)42 and processed separately as described under experimental procedures. Brain homogenate with known protein content was separated by elec-trophoresis and antibody binding to tissue sample and reference standards of known concentration was used to determine B

max

ex vivo, Figure 4. A linear calibration was used to approximate the mass (ng) of HDAC in each brain sample. Using these data, we generated regional distribution plots of each isoform separately (Fig. 4C–E). Qualitatively, our data are consistent with the dis-tribution of mRNA for each isoform provided by the Allen Brain Atlas.43 Of note, HDAC3 protein density is uniform across the

long free and non-specifically bound ligand removal. The affinity range provided that we would be able to measure areas of tissue with reasonably low HDAC1–3 density. We also surmised that the development of an HDAC1–3 ligand could provide a means to map each of the three isoforms selectively (if paired appropriately with a selective inhibi-tor). For example, the use of CI-994 with an HDAC1–2 inhibitor could provide a map of HDAC3 density. Isoform-selective inhibi-tors within this class are indeed known.40,41

With the promising in vitro data in hand, we invested in the synthesis of [3H]CI-994, which was achieved through non-selective hydrogenation (proton exchange) using tritium-gas. The [3H]CI-994 was prepared in sufficiently high specific activ-ity to proceed with initial autoradiography experiments. These experiments had the primary goal of optimization for specific vs. non-specific binding. Our major find-ings through these preliminary experiments were that: (1) incubation indeed needed to be long (hours to days), which necessitated tissue morphology stabilization; (2) specific binding was impacted by over-treatment with the fixative solution and morphology stabilization had to be rigorously controlled; (3) there was essentially no difference observed for samples incu-bated at room temperature vs. 37°C (room temperature was cho-sen because of increased tissue stability over long incubations); (4) washing needed to be accomplished at 4°C and was best when performed using multiple baths; (5) the identity, concentration and pH of the buffer for incubation and washing impacted the ratio of specific to non-specific binding; (6) DMSO must be used to increase the solubility of non-radioactive CI-994 in blocking experiments. In the end, using the optimized procedure outlined in the methods section, we were able to achieve up to 80% spe-cific binding; however, we must note that there can be significant variation from experiment to experiment and thus non-specific binding must be determined in each tissue-sample pair to extract quantitative data.

Determination of specific binding, protein density, and equilibrium binding constant. Using our optimized protocol, we evaluated target saturation by observing total and non-spe-cific binding as a function of [3H]CI-994 concentration (specific binding was calculated from these data). As seen in Figure 2A, regional distribution of [3H]CI-994 was heterogeneous and thus, we analyzed the saturation data both in terms of the whole brain (Fig. 2C) and in terms of individual brain regions (Fig. 3A). We determined that the whole brain B

max (assumed to represent

HDAC1–3 density) was 12.9 picomol·mg−1 protein and the Kd

calculated from saturation was 15.9 nM across the whole brain. In addition to curve fitting analysis, we formatted the data as a Scatchard plot, which was linear, as expected, and provided a

Figure 2. (A) Sagittal and coronal autoradiographic images of total binding and non-specific binding in the rat brain (10 nM [3H]CI-994, 16 h incubation); (B) mean intensity of non-specific binding and total binding of whole rat brain; (C) saturation binding curves for [3H]CI-994 in rat brain: Bmax is 12.9 picomol·mg−1 of protein, Kd is calculated as 15.9 nM and (D) Scatchard plots of [3H]CI-994 binding in rat brain, Bmax is 13.6 picomol·mg−1 of protein, Kd is calculated as 9.59 nM, (n = 6).

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This allowed us to compare Kd deter-

mined in our assay (assumed to be at equi-librium) to K

d derived from the on- and

off-rates. It also provided a means to ana-lyze whether regional uptake was domi-nated by rate differences, which could be indicative of different HDAC forms (e.g., isoform or complex density differences). Figure 5A provides a subset of these data: association curves for the hippocampus and thalamus, which respectively had high and low B

max. The association rates

were different in these two regions; how-ever, we also note a difference in the dis-sociation analysis (Fig. 5B). Of particular note is the remarkably slow kinetics of the benzamide ligand family, which dictates long incubation times to reach equilib-rium binding (several hours, as opposed to only minutes for most ligand receptor interactions). The regional K

d calculated

from these regions are different (9.45 vs. 10.2 nM) and suggest to us that there

may be a regional variation in isoform ratio (or perhaps HDAC-complex identity). Additional studies will need to be performed to fully understand the regional variation in [3H]CI-994 affinity.

Assessment of competitive binding. We envisioned that the development of an autoradiography imaging tool for HDAC would be valuable beyond the characterization of protein density and could be applied to the study of novel ligands using com-petition experiments. As a demonstration of this potential, we validated that the IC

50 for CI-994 competition with [3H]CI-994

matched the Kd as anticipated (Fig. 6A). Interestingly, the IC

50

showed a parallel trend to the kinetic parameters between various brain regions. For example, the IC

50 difference in thalamus and

hippocampus was consistent with the kinetically determined Kd

in those regions. Regional IC50

differences using novel HDAC inhibitors could be used to improve our knowledge of the rela-tionship between in vitro HDAC isoform affinity and in vivo binding (thus site of action) and efficacy. In order to characterize a well-studied and potent HDAC inhibitor, we determined the IC

50 of SAHA using [3H]CI-994 (Fig. 6B). The IC

50 determined

through our assay (1.3–1.5 nM) is consistent with the known potency of SAHA, which is relatively non-selective (as it also targets HDAC6, a class IIb HDAC), but yet highly potent at HDAC1, HDAC2 and HDAC3. We are currently using [3H]CI-994 in competition experiments with HDAC3-selective and HDAC1,2-selective benzamides to explore additional ways to use this autoradiographic tool.

Methods

Chemicals and supplies. CI-994 was provided by the Broad Institute, (Cambridge, MA). Synthesis of [3H]CI-994 (specific activity 47.45 Ci/mmol, radioactivity 1.0 mCi/mL, radiochemi-cal purity > 97.7%) was synthesized by Perkin Elmer. Briefly, [3H]

brain with a density typically twice that of HDAC1 or HDAC2. These data are in effect ‘average’ values within micro-dissected regions and thus variation that is observed as higher resolution is not detected. Also of note are the high densities of HDAC1 and HDAC2 in the cerebellum and the hippocampus.

Using the western-derived densities and the region-specific binding of [3H]CI-994, we plotted correlation plots of individual isoforms vs. [3H]CI-994, as well as all binary combinations. The best correlation, however, was observed by plotting the sum of isoform density by western (Fig. 3B) vs. [3H]CI-994-derived B

max.

These were remarkably well correlated and linear with respect to one-another (R2 = 0.98). B

max values determined by [3H]CI-994

were slightly higher, on average, than those from western-derived values, but this was expected given the extreme differences in the protocols and analyses. From these data, we believe that [3H]CI-994 represents the linear sum of the isoform densities and that the difference in K

d for each isoform (determined in vitro) does

not contribute to the selectivity between isoforms (i.e., the lower K

i of HDAC2 in vitro seems to be inconsequential). Correlations

alone cannot precisely determine the ratio of HDAC1, HDAC2 and HDAC3 binding by [3H]CI-994. We are currently working to block individual isoforms with selective inhibitors to ascribe the precise ratio of isoform binding. For our preliminary analysis, we evaluated the density of ligand binding averaged across large brain regions. However, we note (as seen in Fig. 3C) that sub-structure can be observed. For detailed, high resolution assess-ment of subregions of the hippocampus, long film/phosphor screen exposure times (> 2 mo) will be required; however, with our 7 d exposure data, heterogeneous distribution is noted, for example, in portions of the dentate gyrus.

Determination of ligand binding properties in tissue sec-tions. To rigorously determine the binding parameters of [3H]CI-994 in tissue, we performed kinetic binding experiments.

Figure 3. (A) Bmax of different regions in the rat brain from [3H]CI-994 autoradiography (n = 6) aver-aged over each structure. (B) Correlation plot of ΣHDAC1–3 protein density obtained from western (n = 5) vs. HDACs Bmax obtained from [3H]CI-994 autoradiography (n = 6); R2 is 0.9796. (C) Finer detail within brain regions can be observed for example in the hippocampus where [3H]CI-994 binding is higher in CA3 and the dentate gyrus (DG), and in substructures of the cerebellum.

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over a 4-h period. The recovery of HDAC activity following dilution of the enzyme-[CI-994]-complex is an indication of the reversibility of CI-994. CI-994 off-rates were calculated from dilution progression curve fitting analogously to above.

Western blots. In order to investigate the expression level of class-I HDACs in different brain regions, nine different regions of the mouse brain were micro-dissected. A western blot assay with a known amount of purified human HDACs as standard was used (the affinity of the antibody used should be the same between human and mouse mice HDACs) to calculate the specific amount of HDACs in each brain region. Eight-week old adult WT C57BL/6 mice from Charles River Laboratories International, Inc. were sacrificed by decapitation in accordance with the animal care and use policies from the Subcommittee on Research Animal Care (SRAC) at Massachusetts General Hospital (MGH). The brains were quickly removed and then washed in ice-cold 1X PBS buffer to remove the blood. Water on the surface of the brain was carefully wicked dry using a Kimwipe tissue. The brain was then transferred to a microscope-slide

CI-994 was synthesized by a non-selective catalytic hydrogen-tritium exchange reaction using tritium gas tuner catalyzed by a hydrogen transfer catalyst (Pd/C) under a carrier-free tritium gas atmosphere at 40 psi. The purity and specific radioactivity were determined by HPLC, TLC and mass spectrometry.

For autoradiography, DMSO was obtained from Sigma. TRIS-HCl and PBS buffer were obtained from Fisher Scientific, Inc. Paraformaldehyde (4% PFA) in phosphate-buffered saline was purchased from Boston Bioproducts. Scintillation cocktail, scintillation vials and filter paper were from Perkin Elmer. Slide incubation bath with lids, autoradiography cassette was from Fisher Scientific, Inc. For visualization, cyclone plus storage phosphor system, [3H] supersensitive screens were used (Perkin Elmer).

For western blot, reagent kits for protein characterization assay were obtained from Fisher Scientific, Inc. Primary anti-bodies: rabbit polyclonal to anti-HDAC1 antibody (ab53091), anti-HDAC2-chip grade antibody (ab7029) and anti-HDAC3 antibody (ab16047), each of which bind to mouse, rat and human HDAC, were purchased from Abcam. The secondary antibody (goat anti-rabbit HRP-conjugate) and most of the western blot experimental supplies e.g., criterion 4–15% pre-cast gel, SDS loading, running and transferring buffers, PVDF membrane were purchased from Bio-Rad Laboratories, Inc. Biotin serum albumin was from Fisher Scientific, Inc. Non-fat dry milk was from Labscientific, Inc. Blue loading buffer was from New England BioLabs, Inc. Recombinant HDAC1, 2 and3 purified proteins were purchased from BPS Bioscience, Inc. For imaging, Chemidoc TM XRS+ was used (Bio-Rad Laboratories, Inc.). Micro-dissection tools, forceps, needles, spatulas, razor blade for rats and mice were purchased from Fine Science Tools.

In vitro rate and Kd determination. Slow, tight-binding

kinetics of CI-994 with HDACs 1, 2 and 3 were evaluated by the progression curves in inhibition and dilution experiments.44 Kinetic reactions were assembled in microtiter plate wells by add-ing 1.6 nM, 1.7 nM and 0.5 nM of recombinant, full length, human HDACs 1, 2 and 3 (BPS Biosciences, San Diego), respec-tively, into separate reaction mixtures containing 2 μM HDAC Caliper carboxyfluorescein (FAM)-labeled AcH4K12 peptide substrate (Broad Substrate A) and CI-994 at different concentra-tions. Plates were immediately placed into a LabChip EZ Reader II (Caliper/Perkin Elmer) and the wells were sampled continu-ously throughout a 3-h reaction period. The fluorescent product and substrate were separated based on charge difference between acetylated and deacetylated form, and monitored on the Caliper microfluidic instrument.45 The substrate conversion was calcu-lated with Caliper software. The progression curves at different inhibitor concentrations were analyzed with a nonlinear regres-sion program in Origin 8.5 and fit to the integrated rate equation for slow-binding inhibitors. The off-rate of CI-994 was deter-mined from a dilution experiment. HDAC1 (0.1 μM), HDAC2 (0.1 μM) or HDAC3 (0.05 μM) were incubated with CI-994 at 0.6 μM, 2.5 μM or 0.6 μM respectively for 1 h. The mixtures were then diluted 100-fold into buffer containing 2 μM Broad Substrate A. HDAC-diluted samples were measured continuously

Figure 4. HDAC density assessment in mouse brain using western blot analysis. (A) Western blot analysis of HDAC expression in different regions of mouse brain. Key: Frontal Cortex (FC), Parietal Cortex (PC), Temporal Cortex (TC), Hippocampus (HIP), Thalamus (TH), Striatum (ST), Hypothalamus (HYP), Olfactory Bulb (OB) and Cerebellum (CER). (B) Lin-earity of HDAC standard analysis by optical densitometry using imageJ analyzer; blue = HDAC1, black = HDAC2, orange = HDAC3. (C) HDAC1 protein density in regions of mouse brain. (D) HDAC2 protein density in regions of mouse brain. (E) HDAC3 protein density in different regions of mouse brains (n = 5).

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Protein preparation. After weighing the collected tissue sections, each tissue was homogenized by treating with 300 μL of lys-ing buffer (2 × RIPA) (25 mM TRIS-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS). A homogenization pestle was used followed by vortex, incubation and centrifugation to remove insoluble materi-als. The supernatant was collected and frozen at −20°C; the debris was discarded. The fro-zen pellets of mouse brain homogenate were thawed on the day of assay. HDAC standard (reference) proteins and the clarified protein supernatant concentration were colorimetri-cally determined (at 562 nM by BCA protein assay for total protein concentration by follow-ing the protocol provided by the Pierce BCA protein assay kit).47 HDAC1, HDAC2 and HDAC3 levels in each of nine brain regions were analyzed by western blot in triplicate.

Proteins (15 μg/lane) were separated by SDS-PAGE and then transferred onto PVDF membrane. The membrane was blocked with 5% of non-fat dry milk in 1× Tris-buffered saline contain-ing 0.5% tween-20 for 1 h. The blots were incubated with their respective primary antibody (1:10,000) for 16 h at 4°C. Blots were visualized after incubation with an anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:50,000) for 1 h, then developed with Bio-Rad Chem. luminescence. The amount of each HDAC in the different regions of mouse brains was determined and normalized to loaded protein mass. Quantitative analysis was performed densitometrically using an Image J ana-lyzer (NIH) and data are reported in pmol·mg−1 protein (not to be confused with pmol·mg−1 tissue).

Plasma and brain homogenate binding assays. Brain bind-ing assays were performed by Sai Advantium Pharma Limited (Hinjewadi). Briefly, rapid equilibrium dialysis was performed with a rapid equilibrium dialysis (RED) device containing dialy-sis membrane with a molecular weight cut-off of 8,000 Daltons. Each dialysis insert contains two chambers. The red chamber is for plasma or brain homogenate while the white chamber is for buffer. A 200 μL aliquot of CI-994 in brain homogenate (tripli-cates) was added to the red chamber of dialysis inserts. A 350 μL aliquot of dialysis buffer was added to the buffer chamber of the inserts. Carbamazepine was used as positive control for brain tis-sue binding. After sealing the RED device with an adhesive film, dialysis was done at 37°C with shaking at 100 rpm for 4 h. A 50 μL aliquot of CI-994 and positive controls was separately added to 0.5 mL Eppendorf tubes. Two aliquots were frozen immediately (0 min sample). Two other aliquots were incubated at 37°C for 4 h along with the RED device. Following dialysis, an aliquot of 50 μL was removed from each well (brain homogenate and buffer) and diluted with equal volume of opposite matrix to nullify the matrix effect. Similarly, buffer was added to recovery and stability samples. An aliquot of 100 μL was submitted for LC-MS/MS analysis. A 25 μL aliquot of the carbamazepine and CI-994 were treated with 100 μL of MeCN containing internal

sitting on a bed of dry ice, loosely covered with aluminum foil and allowed to freeze for 10 min. Before sectioning the brain, it was rinsed with PBS buffer at 4°C. The brain was mounted on the microscope slide and cut in coronal slabs from olfactory bulb to cerebellum with thickness of 1 cm and the slabs were arranged in the Leica dissection microscope in sequence. Slabs were then micro-dissected in coronal orientation at room temperature as quickly as possible. Nine different regions of the mouse brain were dissected which included frontal cortex, parietal cortex, temporal cortex, hippocampus, thalamus, striatum, hypothala-mus, olfactory bulb and cerebellum.46 The dissected tissues were placed in Eppendorf tubes and quickly transferred to dry ice. For long-term storage they were later transferred to a −80°C freezer.

Figure 6. (A) CI-994 self-competition curves (n = 6) in the whole brain slice, hippocampus and thalamus of the rat brain, (left to right); (B) SAHA competition curves in the corresponding rat brain tissues, (n = 6).

Figure 5. [3H]CI-994 Binding Kinetics. The observed on and off rates were calculated from association (A) and dissociation (B) curves using the one-phase exponential association and decay equations. Saturation occurred after 24 h. Half saturation was reached around 3 h. Dissociation was slow in both high and low binding regions with a t1/2 around 8 h. Kd can be calculated from the on and off rates: Kd = Koff/Kon·Kd (HIP) is 9.45 nM; and Kd (TH) is 10.2 nM, (n = 6), which is consistent with the Kd obtained from saturation binding value.

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Cyclone Phosphor Imager (Perkin Elmer) was used to record and visualize the phosphor-screen data.

Binding optimization experiments. Consecutive tissue sec-tions in coronal and sagittal orientation throughout the rat brain (mounted and fixed as described above) were sequentially num-bered divided into two groups: odd numbered tissue sections were used for non-specific binding and even numbered tissue sections were used for total binding. Odd numbered tissue section were treated with 100 μM cold CI-994 plus 10 nM [3H]CI-994 in 5% DMSO and 10 mM TRIS-HCl buffer; even numbered tis-sue section were treated with 10 nM [3H]CI-994 in 5% DMSO and 10 mM TRIS-HCl buffer. Optimization of incubation time, temperature and the rinsing protocol was achieved systemically and led to standard set of conditions above.

Saturation binding assay. Alternating adjacent sections (as above) were incubated with increasing concentrations of radioli-gand [3H]CI-994 from 1 nM to 100 nM, either with or without excess 100 μM unlabeled CI-994. Saturation experiments were simultaneously performed on different anatomical regions of the rat brain: the frontal cortex, parietal cortex, temporal cortex, stri-atum, hippocampus, thalamus, hypothalamus and cerebellum in coronal and sagittal planes, respectively. Sections from each indi-vidual brain were exposed to the same phosphor screen film. A calibration standard was used to determine the efficiency differ-ence between each of the phosphor screens used. B

max and K

d were

calculated by curve fitting and through a Scatchard plot analysis.Dissociation and association kinetics experiments. Thaw

mounted tissue sections in both coronal and sagittal orientations were treated with 10 nM [3H]CI-994 either with or without 100 μM unlabeled CI-994 according to the general method for 16 h. After transferring samples to the second washing bath (time zero), samples were removed from the baths at increasing time intervals from 1 to 48 h (according to Fig. 5B) to determine dis-sociation of both total and non-specifically bound [3H]CI-994. The rate of dissociation for specific bound [3H]CI-994 was calcu-lated from each time-point pair of sample (total − non-specific). To determine association rate, the incubation time with [3H]CI-994 was varied from 1 to 48 h (washing was held constant at 16 h). Specific binding association rate was determined in an analogous manner. Curve-fitting in GraphPad Prism was used to extract k

off (dissociation rate), which was used in the determi-

nation of kon

(association rate). Rate-derived Kd was determined

using these rate constants.Self- and SAHA-competitive (IC

50) binding experiments.

Two parallel experiments were designed for competitive binding assay: CI-994 (self) and SAHA competition binding experiments. Thaw mounted tissue sections in both coronal and sagittal ori-entations were treated with unlabeled CI-994 or SAHA in a con-centration range from 100 pM to 100 μM (according to Fig. 6) mixed with 10 nM [3H]CI-994 according to the general method. Competition curves were obtained by plotting total binding of [3H]CI-994 against the various (log) concentrations of the com-peting ligand (i.e., CI-994 or SAHA). IC

50 was determined by

curve fitting in GraphPad Prism.Quantitative data analysis (calibration to protein density).

All autoradiography data were analyzed using densitometry using

standards (caffeine 200 ng/mL in MeCN) and vortexed for 5 min. The samples were centrifuged at 15,000 rpm at 4°C for 10 min and 100 μL of supernatant was submitted for LC-MS/MS analysis. Samples were monitored for parent compound in MRM mode using LC-MS/MS.

Animal procedures and tissue preparation for autoradiogra-phy. The experimental procedures were performed according to a protocol approved by the Subcommittee on Research Animal Care (SRAC) at Massachusetts General Hospital (MGH). Brain tissue was obtained by decapitating 8-week adult male Sprague-Dawley rats (150–200 g), in accordance with MGH SRAC’s animal care and use policies. The animals were sacrificed by intraperitoneal injection of pentobarbital. The whole brains were removed and obtained rapidly, after a quick wash in cold 1XPBS; the brains were rapidly frozen by placing the brain on a microscope slide situated on a bed of dry ice. Once frozen, each brain was mounted for cryostat sectioning. Coronal and sagit-tal sections (20 μm) were consecutively cut and thaw mounted on gelatin-coated slides, which were from Fisher Scientific, Inc. Slides were stored at −80°C, and based on our experience, should be used no more than two weeks after initial storage.

General experimental method. For most experiments, two parallel experiments were performed. One bath-series was used to measure total binding; and the other was used to determine non-specific binding. The slide-mounted consecutive tissue sec-tions were first fixed with 4% of paraformaldehyde and 2% of ethanol in 1XPBS buffer for 1 h at 4°C to stabilize the morphol-ogy of the tissues. The tissue sections were then pre-incubated in 10 mM TRIS-HCl buffer, pH 7.5 for 10 min at room tempera-ture in an attempt to dissociate endogenous ligand and remove any residue from cryostat slide mounting. A second bath-set (2 h at room temperature) was used to pre-saturate the ligand targets (for non-specific binding determination); a vehicle only bath was used for parity in the total binding experiments. Non-specific binding was determined using adjacent sections with 100 μM non-radioactive CI-994 in 5% DMSO and 10 mM TRIS-HCl buffer. (CI-994 should be first dissolved in DMSO then diluted to the concentration as needed by adding the TRIS-HCl buf-fer). A third bath-set was used for the [3H]CI-994 incubation. The total binding bath consisted of 10 nM [3H]CI-994 in 5% DMSO and 10 mM TRIS-HCl buffer; the non-specific binding bath consisted of 10 nM [3H]CI-994 and 100 μM non-radioac-tive CI-994 in 5% DMSO and 10 mM TRIS-HCl buffer. Each incubation bath was covered with a lid to maintain the humid-ity and incubation was performed for 16 h at room temperature (unless otherwise noted). Following incubation, the slides were transferred into two sequential baths in an attempt to remove free and non-specifically bound [3H]CI-994 (10 mM Tris-Hcl buffer, pH 7.5 at 4°C): the first rinse was performed for 30 sec and the next for 16 h. After washing the sections in buffer, a deionized water bath (4°C) was used to remove buffer salts. The sections were then dried at reduced pressure (in a vacuum oven) at room temperature for 24 h. Once dry, the microscope slide were placed tissue-side down on tritium-sensitive phosphor screens, which were subsequently sealed in autoradiography cassettes and moved to a leaded storage box for 1 week, unless otherwise stated. A

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and it will afford an option for measuring (at least ex vivo) the change in HDAC1–3 expression as a function of normal aging processes and biological diversity (e.g., sex). [3H]CI-994 can be used to characterize HDAC1–3 response to environmental inputs and will be valuable for exploring the relationships that exist between disease state and progression with respect to HDAC density. These studies can be accomplished on preserved human post mortem tissue provided that fixation of the tissue is mild to moderate. In preclinical settings, [3H]CI-994 may be also valu-able for exploring HDACi therapy response and mechanism. In preliminary experiments, we have been successful in using [3H]CI-994 to study HDAC1–3 in other tissues (such as the heart) and found that the data provided herein for the brain are gener-ally applicable to other organs.

Ultimately, we envision the development of probes to mea-sure HDAC density in vivo and [3H]CI-994 will be important for prescreening compounds to prioritize for PET imaging. As an in vivo imaging ligand itself, we have used [11C]CI-994 and have determined that the in vivo blood-brain-barrier penetration of CI-994 is not sufficient for PET imaging in non-human pri-mates; however, we are still characterizing its binding properties in other organs. We suspect that faster binding kinetic will be required for in vivo applications and are pursuing other classes of HDAC inhibitors.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors would like to thank Drs Jinsheng Yang and Wei Zhao for technical support in analysis of the autoradiography data, Dr Chao Wei’s lab for mouse brain tissue, Drs Changning Wang and Frederick ‘Al’ Schroeder for scientific discussion and the NIH for financial support (5R01DA030321, P30DA028800, and R01DA028301).

ImageJ Analyzer software (NIH). Specific binding measured in optical density (OD, non-normalized) was obtained by sub-tracting paired ROIs of non-specific binding and total binding. The non-specific and total binding ODs had been corrected for background signal using a local non-tissue region of the slide area analyzed. All pairs analyzed through subtraction in this way were recorded adjacently on a phosphor screen. Each screen (and data set) was calibrated from OD to nCi·mg−1 plastic standard using a [3H]-radioactive standard from American Radiolabeled Chemical, Inc. The standard samples were analogously treated in terms of ROI size and back subtraction and resulted in a calibration curve fit to a second order polynomial with R2 = 0.99 in Microsoft Excel. The polynomial equation was used for the conversion of OD to nCi·mg−1 plastic standard. To determine the relationship between nCi·mg−1 plastic standard and nCi·mg−1 protein, eight tissue sam-ples of known size (2.1 cm × 1.0 cm) from cryostat sectioning were cut and weighed. These samples were homogenized as out-lined in the western blot analysis details to quantify the protein content in the samples. Using the known tissue section volume, mass, and protein density values that were determined, nCi·mg−1 plastic standard values were converted to nCi·mg−1 protein (for our analyses the conversion factor between these two values was 4.14). To convert from nCi to fmol, the specific radioactivity of [3H]CI-994 was used. Thus, the final data were represented in fmol of CI-994 specific binding·mg−1 total protein. Assuming that all specific binding occurs at HDAC1–3, this value represents the total density of HDAC1–3 in the tissue sections.

Conclusion and Outlook

[3H]CI-994 is now the first validated tool for imaging class I histone deacetylases by autoradiography and can now be used for probing regional variation in HDAC1–3 density in many experiment designs. For example, [3H]CI-994 will be valuable for studying the affinity of novel HDAC inhibitors in tissue sections

References1. Clayton AL, Hazzalin CA, Mahadevan LC. Enhanced

histone acetylation and transcription: a dynamic per-spective. Mol Cell 2006; 23:289-96; PMID:16885019; http://dx.doi.org/10.1016/j.molcel.2006.06.017

2. Cohen I, Poreba E, Kamieniarz K, Schneider R. Histone modifiers in cancer: friends or foes? Genes Cancer 2011; 2:631-47; PMID:21941619; http://dx.doi.org/10.1177/1947601911417176

3. Spencer CA, Kruhlak MJ, Jenkins HL, Sun X, Bazett-Jones DP. Mitotic transcription repression in vivo in the absence of nucleosomal chromatin condensation. J Cell Biol 2000; 150:13-26; PMID:10893252; http://dx.doi.org/10.1083/jcb.150.1.13

4. Haberland M, Montgomery RL, Olson EN. The many roles of histone deacetylases in development and physiology: implications for disease and therapy. Nat Rev Genet 2009; 10:32-42; PMID:19065135; http://dx.doi.org/10.1038/nrg2485

5. Hendricks JA, Keliher EJ, Marinelli B, Reiner T, Weissleder R, Mazitschek R. In vivo PET imaging of histone deacetylases by 18F-suberoylanilide hydroxam-ic acid (18F-SAHA). J Med Chem 2011; 54:5576-82; PMID:21721525; http://dx.doi.org/10.1021/jm200620f

6. Wilting RH, Dannenberg JH. Epigenetic mecha-nisms in tumorigenesis, tumor cell heterogeneity and drug resistance. Drug Resist Updat 2012; 15:21-38; PMID:22356866; http://dx.doi.org/10.1016/j.drup.2012.01.008

7. Chavan AV, Somani RR. HDAC inhibitors - new generation of target specific treatment. Mini Rev Med Chem 2010; 10:1263-76; PMID:20701588; http://dx.doi.org/10.2174/13895575110091263

8. Shein NA, Shohami E. Histone deacetylase inhibitors as therapeutic agents for acute central nervous system injuries. Mol Med 2011; 17:448-56; PMID:21274503; http://dx.doi.org/10.2119/molmed.2011.00038

9. Jia H, Kast RJ, Steffan JS, Thomas EA. Selective his-tone deacetylase (HDAC) inhibition imparts beneficial effects in Huntington’s disease mice: implications for the ubiquitin-proteasomal and autophagy systems. Hum Mol Genet 2012; 21:5280-93; PMID:22965876; http://dx.doi.org/10.1093/hmg/dds379

10. Schmalbach S, Petri S. Histone deacetylation and motor neuron degeneration. CNS Neurol Disord Drug Targets 2010; 9:279-84; PMID:20406183; http://dx.doi.org/10.2174/187152710791292684

11. Echaniz-Laguna A, Bousiges O, Loeffler JP, Boutillier AL. Histone deacetylase inhibitors: therapeutic agents and research tools for deciphering motor neuron diseases. Curr Med Chem 2008; 15:1263-73; PMID:18537606; http://dx.doi.org/10.2174/092986708784534974

12. Tremolizzo L, Rodriguez-Menendez V, Sala G, Di Francesco JC, Ferrarese C. Valproate and HDAC inhi-bition: a new epigenetic strategy to mitigate phenotypic severity in ALS? Amyotroph Lateral Scler Other Motor Neuron Disord 2005; 6:185-6; PMID:16183561; http://dx.doi.org/10.1080/14660820510033614

13. Grayson DR, Kundakovic M, Sharma RP. Is there a future for histone deacetylase inhibitors in the phar-macotherapy of psychiatric disorders? Mol Pharmacol 2010; 77:126-35; PMID:19917878; http://dx.doi.org/10.1124/mol.109.061333

14. Kazantsev AG, Thompson LM. Therapeutic appli-cation of histone deacetylase inhibitors for central nervous system disorders. Nat Rev Drug Discov 2008; 7:854-68; PMID:18827828; http://dx.doi.org/10.1038/nrd2681

15. Fass DM, Schroeder FA, Perlis RH, Haggarty SJ. Epigenetic mechanisms in mood disorders: Targeting neuroplasticity. Neuroscience 2013; •••:S0306-4522; PMID:23376737

16. Jawerka M, Colak D, Dimou L, Spiller C, Lagger S, Montgomery RL, et al. The specific role of histone deacetylase 2 in adult neurogenesis. Neuron Glia Biol 2010; 6:93-107; PMID:20388229; http://dx.doi.org/10.1017/S1740925X10000049

Dow

nloa

ded

by [

Har

vard

Lib

rary

] at

07:

57 1

9 D

ecem

ber

2014

©20

13 L

ande

s B

iosc

ienc

e. D

o no

t dis

tribu

te.


36. Chou DH, Holson EB, Wagner FF, Tang AJ, Maglathlin RL, Lewis TA, et al. Inhibition of histone deacetylase 3 protects beta cells from cytokine-induced apoptosis. Chem Biol 2012; 19:669-73; PMID:22726680; http://dx.doi.org/10.1016/j.chembiol.2012.05.010

37. Li-Huei Tsai JSG, Haggarty SJ, Holson E, Wagner F, Graeff J. Use of CI-994 and dinaline for the treatment of memory/cognition and anxiety disorders. 2010.

39. Bradner JE, Mak R, Tanguturi SK, Mazitschek R, Haggarty SJ, Ross K, et al. Chemical genetic strat-egy identifies histone deacetylase 1 (HDAC1) and HDAC2 as therapeutic targets in sickle cell dis-ease. Proc Natl Acad Sci U S A 2010; 107:12617-22; PMID:20616024; http://dx.doi.org/10.1073/pnas.1006774107

40. Cavasin MA, Demos-Davies K, Horn TR, Walker LA, Lemon DD, Birdsey N, et al. Selective class I histone deacetylase inhibition suppresses hypoxia-induced cardiopulmonary remodeling through an antiproliferative mechanism. Circ Res 2012; 110:739-48; PMID:22282194; http://dx.doi.org/10.1161/CIRCRESAHA.111.258426

41. Hu E, Dul E, Sung CM, Chen Z, Kirkpatrick R, Zhang GF, et al. Identification of novel isoform-selective inhib-itors within class I histone deacetylases. J Pharmacol Exp Ther 2003; 307:720-8; PMID:12975486; http://dx.doi.org/10.1124/jpet.103.055541

42. Glowinski J, Axelrod J. Effects of drugs on the disposi-tion of H-3-norepinephrine in the rat brain. Pharmacol Rev 1966; 18:775-85; PMID:5904189

43. Allen Brain Atlas. [cited 2012; Available from: http://www.brain-map.org/.

44. Zhang Y, Holson EB, Wagner FF. inventors. The Broad Institute, Inc., assignee. Fluorescent substrates for determining lysine modifying enzyme activity. United States patent US WO2013067391. 2013 Oct 05.

45. Holson EW, Weïwer M, Zhang YL. inventors. The Broad Institute, Inc., assignee. Inhibitors of histone deacetylases. United States patent US WO2012149540. 2012 Jan 11.

46. Li KW, ed. Neuroproteomics (Neuromethods). Dissection of Rodent Brain Regions, ed. S. Spijker. Vol. 57. 2011. 13-26.

47. Tyllianakis PE, Kakabakos SE, Evangelatos GP, Ithakissios DS. Direct colorimetric determination of solid-supported functional groups and ligands using bicinchoninic acid. Anal Biochem 1994; 219:335-40; PMID:8080090; http://dx.doi.org/10.1006/abio.1994.1273

27. Hooker JM, Kim SW, Alexoff D, Xu Y, Shea C, Reid A, et al. Histone deacetylase inhibitor, MS-275, exhib-its poor brain penetration: PK studies of [C]MS-275 using Positron Emission Tomography. ACS Chem Neurosci 2010; 1:65-73; PMID:20657706; http://dx.doi.org/10.1021/cn9000268

28. Reid AE, Hooker J, Shumay E, Logan J, Shea C, Kim SW, et al. Evaluation of 6-([(18)F]fluoroacetamido)-1-hexanoicanilide for PET imaging of histone deacety-lase in the baboon brain. Nucl Med Biol 2009; 36:247-58; PMID:19324270; http://dx.doi.org/10.1016/j.nucmedbio.2008.12.005

29. Undevia SD, Kindler HL, Janisch L, Olson SC, Schilsky RL, Vogelzang NJ, et al. A phase I study of the oral combination of CI-994, a putative histone deacetylase inhibitor, and capecitabine. Ann Oncol 2004; 15:1705-11; PMID:15520075; http://dx.doi.org/10.1093/annonc/mdh438

30. Pauer LR, Olivares J, Cunningham C, Williams A, Grove W, Kraker A, et al. Phase I study of oral CI-994 in combination with carboplatin and paclitaxel in the treatment of patients with advanced solid tumors. Cancer Invest 2004; 22:886-96; PMID:15641487; http://dx.doi.org/10.1081/CNV-200039852

31. Perabo FG, Müller SC. New agents for treatment of advanced transitional cell carcinoma. Ann Oncol 2007; 18:835-43; PMID:17018703; http://dx.doi.org/10.1093/annonc/mdl331

32. Gridelli C, Rossi A, Maione P. The potential role of histone deacetylase inhibitors in the treatment of non-small-cell lung cancer. Crit Rev Oncol Hematol 2008; 68:29-36; PMID:18424067; http://dx.doi.org/10.1016/j.critrevonc.2008.03.002

33. Ropero S, Esteller M. The role of histone deacetylases (HDACs) in human cancer. Mol Oncol 2007; 1:19-25; PMID:19383284; http://dx.doi.org/10.1016/j.molonc.2007.01.001

34. Hubeek I, Comijn EM, Van der Wilt CL, Merriman RL, Padron JM, Kaspers GJ, et al. CI-994 (N-acetyl-dinaline) in combination with conventional anti-cancer agents is effective against acute myeloid leukemia in vitro and in vivo. Oncol Rep 2008; 19:1517-23; PMID:18497959

35. Richards DA, Boehm KA, Waterhouse DM, Wagener DJ, Krishnamurthi SS, Rosemurgy A, et al. Gemcitabine plus CI-994 offers no advantage over gemcitabine alone in the treatment of patients with advanced pancreatic cancer: results of a phase II randomized, double-blind, placebo-controlled, multicenter study. Ann Oncol 2006; 17:1096-102; PMID:16641168; http://dx.doi.org/10.1093/annonc/mdl081

17. Delcuve GP, Khan DH, Davie JR. Roles of his-tone deacetylases in epigenetic regulation: emerg-ing paradigms from studies with inhibitors. Clin Epigenetics 2012; 4:5; PMID:22414492; http://dx.doi.org/10.1186/1868-7083-4-5

18. Gräff J, Kim D, Dobbin MM, Tsai LH. Epigenetic regulation of gene expression in physiological and path-ological brain processes. Physiol Rev 2011; 91:603-49; PMID:21527733; http://dx.doi.org/10.1152/phys-rev.00012.2010

19. Guan JS, Haggarty SJ, Giacometti E, Dannenberg JH, Joseph N, Gao J, et al. HDAC2 negatively regulates memory formation and synaptic plasticity. Nature 2009; 459:55-60; PMID:19424149; http://dx.doi.org/10.1038/nature07925

20. Biermann J, Boyle J, Pielen A, Lagrèze WA. Histone deacetylase inhibitors sodium butyrate and valproic acid delay spontaneous cell death in purified rat retinal ganglion cells. Mol Vis 2011; 17:395-403; PMID:21311741

21. Montgomery RL, Hsieh J, Barbosa AC, Richardson JA, Olson EN. Histone deacetylases 1 and 2 control the progression of neural precursors to neurons dur-ing brain development. Proc Natl Acad Sci U S A 2009; 106:7876-81; PMID:19380719; http://dx.doi.org/10.1073/pnas.0902750106

22. Cardinale JP, Sriramula S, Pariaut R, Guggilam A, Mariappan N, Elks CM, et al. HDAC inhi-bition attenuates inflammatory, hypertrophic, and hypertensive responses in spontaneously hyper-tensive rats. Hypertension 2010; 56:437-44; PMID:20679181; http://dx.doi.org/10.1161/HYPERTENSIONAHA.110.154567

23. Chen Y, Wang H, Yoon SO, Xu X, Hottiger MO, Svaren J, et al. HDAC-mediated deacetylation of NF-κB is critical for Schwann cell myelination. Nat Neurosci 2011; 14:437-41; PMID:21423191; http://dx.doi.org/10.1038/nn.2780

24. Miller RH. Unwrapping HDAC1 and HDAC2 func-tions in Schwann cell myelination. Nat Neurosci 2011; 14:401-3; PMID:21445062; http://dx.doi.org/10.1038/nn.2788

25. MacDonald JL, Roskams AJ. Histone deacetylases 1 and 2 are expressed at distinct stages of neuro-glial development. Dev Dyn 2008; 237:2256-67; PMID:18651664; http://dx.doi.org/10.1002/dvdy.21626

26. Broide RS, Redwine JM, Aftahi N, Young W, Bloom FE, Winrow CJ. Distribution of histone deacetylases 1-11 in the rat brain. J Mol Neurosci 2007; 31:47-58; PMID:17416969; http://dx.doi.org/10.1007/BF02686117

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class i hdac imaging using [3h]ci-994 autoradiography

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