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Histone Deacetylase (HDAC) Inhibitor Kinetic Rate Constants Correlate with Cellular Histone Acetylation but Not Transcription and Cell Viability

Open AccessPublished:July 29, 2013DOI:https://doi.org/10.1074/jbc.M113.490706
      Histone deacetylases (HDACs) are critical in the control of gene expression, and dysregulation of their activity has been implicated in a broad range of diseases, including cancer, cardiovascular, and neurological diseases. HDAC inhibitors (HDACi) employing different zinc chelating functionalities such as hydroxamic acids and benzamides have shown promising results in cancer therapy. Although it has also been suggested that HDACi with increased isozyme selectivity and potency may broaden their clinical utility and minimize side effects, the translation of this idea to the clinic remains to be investigated. Moreover, a detailed understanding of how HDACi with different pharmacological properties affect biological functions in vitro and in vivo is still missing. Here, we show that a panel of benzamide-containing HDACi are slow tight-binding inhibitors with long residence times unlike the hydroxamate-containing HDACi vorinostat and trichostatin-A. Characterization of changes in H2BK5 and H4K14 acetylation following HDACi treatment in the neuroblastoma cell line SH-SY5Y revealed that the timing and magnitude of histone acetylation mirrored both the association and dissociation kinetic rates of the inhibitors. In contrast, cell viability and microarray gene expression analysis indicated that cell death induction and changes in transcriptional regulation do not correlate with the dissociation kinetic rates of the HDACi. Therefore, our study suggests that determining how the selective and kinetic inhibition properties of HDACi affect cell function will help to evaluate their therapeutic utility.
      Background: The effect of HDAC inhibitor kinetic properties on biological function is currently unknown.
      Results: The kinetic rate constants of HDAC inhibitors differentially affect histone acetylation, cell viability, and gene expression.
      Conclusion: Evaluating HDAC inhibitor properties using histone acetylation is not predictive of their function on cellular activity.
      Significance: Characterizing the biological effect of different HDAC inhibitors will help to evaluate their clinical utility.

      Introduction

      Over the last decade, increasing evidence has demonstrated that the ability of an organism to respond to its environment resides largely in its capacity to modulate gene expression programs by altering DNA and chromatin structure. Chromatin remodeling by the post-translational modification of histones is thought to be a central mechanism for the epigenetic regulation of gene expression (
      • Shahbazian M.D.
      • Grunstein M.
      Functions of site-specific histone acetylation and deacetylation.
      ). Although several of these modifications such as phosphorylation, ubiquitination, and ADP-ribosylation have been previously identified, the best characterized and most abundant modifications are methylation and acetylation (
      • Kouzarides T.
      Chromatin modifications and their function.
      ). Histone acetylation is regulated by two enzyme families that are “writers” and “erasers” of these modifications. Histone acetyltransferases catalyze the transfer of an acetyl group to histones and favor chromatin opening and gene expression, whereas histone deacetylases (HDACs)
      The abbreviations used are: HDAC, histone deacetylase; HDACi, HDAC inhibitor; SAHA, vorinostat; TSA, trichostatin-A; TCEP, tris(2-carboxyethyl)phosphine; CHES, 2-(cyclohexylamino)ethanesulfonic acid.
      remove acetyl groups and promote transcriptionally silent chromatin (
      • Grunstein M.
      Histone acetylation in chromatin structure and transcription.
      ,
      • Kuo M.H.
      • Allis C.D.
      Roles of histone acetyltransferases and deacetylases in gene regulation.
      ). Eighteen HDACs have been identified in the human genome and can be divided into two main groups, the zinc-dependent HDACs (class I, II, and IV) and the NAD+-dependent sirtuins (class III). HDACs have also been divided into different classes according to phylogenic sequence and function, with HDAC1–3 and -8 belonging to class I, HDAC4, -5, -7, and -9 to class IIa, HDAC6 and HDAC10 to class IIb, and HDAC11 as the only member of class IV. Sirtuins 1–7 form class III (
      • Allis C.D.
      • Berger S.L.
      • Cote J.
      • Dent S.
      • Jenuwien T.
      • Kouzarides T.
      • Pillus L.
      • Reinberg D.
      • Shi Y.
      • Shiekhattar R.
      • Shilatifard A.
      • Workman J.
      • Zhang Y.
      New nomenclature for chromatin-modifying enzymes.
      ).
      Although it is recognized that histone acetylation plays a central role in controlling gene expression, mounting evidence indicates that dysregulation of histone acetylation is a common feature associated with a wide range of disorders, including cardiovascular diseases, cancer, inflammation, metabolic disorders, and neurological diseases (
      • Shakespear M.R.
      • Halili M.A.
      • Irvine K.M.
      • Fairlie D.P.
      • Sweet M.J.
      Histone deacetylases as regulators of inflammation and immunity.
      ,
      • Arrowsmith C.H.
      • Bountra C.
      • Fish P.V.
      • Lee K.
      • Schapira M.
      Epigenetic protein families: a new frontier for drug discovery.
      ,
      • McKinsey T.A.
      Therapeutic potential for HDAC inhibitors in the heart.
      ,
      • Gräff J.
      • Kim D.
      • Dobbin M.M.
      • Tsai L.H.
      Epigenetic regulation of gene expression in physiological and pathological brain processes.
      ,
      • Wiech N.L.
      • Fisher J.F.
      • Helquist P.
      • Wiest O.
      Inhibition of histone deacetylases: A pharmacological approach to the treatment of non-cancer disorders.
      ). Indeed, alteration of histone acetylation levels have been observed in multiple forms of cancer (
      • Baylin S.B.
      • Jones P.A.
      A decade of exploring the cancer epigenome—biological and translational implications.
      ). One of the best examples is the aberrant activity of class I HDACs in t(8;21) acute myeloid leukemia, which silences AML1 target genes involved in hematopoietic stem cell differentiation and promotion of leukemogenesis (
      • Erickson P.
      • Gao J.
      • Chang K.S.
      • Look T.
      • Whisenant E.
      • Raimondi S.
      • Lasher R.
      • Trujillo J.
      • Rowley J.
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      Identification of breakpoints in t(8;21) acute myelogenous leukemia and isolation of a fusion transcript, AML1/ETO, with similarity to Drosophila segmentation gene, runt.
      ,
      • Hug B.A.
      • Lazar M.A.
      ETO interacting proteins.
      ). Importantly, blocking HDAC enzymatic activity with small molecule HDAC inhibitors has been shown to reduce malignancies in vitro and in vivo by blocking the cell cycle and inducing apoptosis (
      • Khan O.
      • La Thangue N.B.
      HDAC inhibitors in cancer biology: emerging mechanisms and clinical applications.
      ). Moreover, several HDACi, including vorinostat (SAHA), entinostat (MS-275), and valproic acid, have been tested in clinical trials with SAHA and rhomidepsin already approved for use in cutaneous T-cell lymphoma patients.
      Recent work has implicated disruption of histone acetylation in neurodegenerative diseases of aging such as amyotrophic lateral sclerosis and Alzheimer disease and in psychiatric conditions such as schizophrenia (
      • Shahbazian M.D.
      • Grunstein M.
      Functions of site-specific histone acetylation and deacetylation.
      ,
      • Gräff J.
      • Kim D.
      • Dobbin M.M.
      • Tsai L.H.
      Epigenetic regulation of gene expression in physiological and pathological brain processes.
      ,
      • Guidotti A.
      • Auta J.
      • Chen Y.
      • Davis J.M.
      • Dong E.
      • Gavin D.P.
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      • Matrisciano F.
      • Pinna G.
      • Satta R.
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      • Tremolizzo L.
      • Tueting P.
      Epigenetic GABAergic targets in schizophrenia and bipolar disorder.
      ,
      • Chuang D.-M.
      • Leng Y.
      • Marinova Z.
      • Kim H.J.
      • Chiu C.-T.
      Multiple roles of HDAC inhibition in neurodegenerative conditions.
      ). In particular, increased levels of HDAC2 have been described in the spinal cord of human patients with amyotrophic lateral sclerosis as well as in the hippocampus of patients suffering from Alzheimer disease, although HDAC2 levels have been found to be decreased in the nucleus accumbens of depressed patients studied postmortem (
      • Kouzarides T.
      Chromatin modifications and their function.
      ,
      • Gräff J.
      • Rei D.
      • Guan J.-S.
      • Wang W.-Y.
      • Seo J.
      • Hennig K.M.
      • Nieland T.J.
      • Fass D.M.
      • Kao P.F.
      • Kahn M.
      • Su S.C.
      • Samiei A.
      • Joseph N.
      • Haggarty S.J.
      • Delalle I.
      • Tsai L.-H.
      An epigenetic blockade of cognitive functions in the neurodegenerating brain.
      ,
      • Janssen C.
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      • Sarlette A.
      • Dengler R.
      • Petri S.
      Differential histone deacetylase mRNA expression patterns in amyotrophic lateral sclerosis.
      ,
      • Covington 3rd, H.E.
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      • Mouzon E.
      • Neve R.L.
      • Tamminga C.A.
      • Nestler E.J.
      A role for repressive histone methylation in cocaine-induced vulnerability to stress.
      ). Moreover, HDACi have been shown to rescue neurological symptoms in mouse models of amyotrophic lateral sclerosis, Alzheimer disease, and depression, suggesting that HDACi could be used in the treatment of chronic neurological diseases (
      • Grunstein M.
      Histone acetylation in chromatin structure and transcription.
      ,
      • Kuo M.H.
      • Allis C.D.
      Roles of histone acetyltransferases and deacetylases in gene regulation.
      ,
      • Ryu H.
      • Smith K.
      • Camelo S.I.
      • Carreras I.
      • Lee J.
      • Iglesias A.H.
      • Dangond F.
      • Cormier K.A.
      • Cudkowicz M.E.
      • Brown Jr., R.H.
      • Ferrante R.J.
      Sodium phenylbutyrate prolongs survival and regulates expression of anti-apoptotic genes in transgenic amyotrophic lateral sclerosis mice.
      ,
      • Govindarajan N.
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      • Walter J.
      • Sananbenesi F.
      • Fischer A.
      Sodium butyrate improves memory function in an Alzheimer's disease mouse model when administered at an advanced stage of disease progression.
      ,
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      • Frechilla D.
      • Del Río J.
      • García-Osta A.
      Phenylbutyrate ameliorates cognitive deficit and reduces tau pathology in an Alzheimer's disease mouse model.
      ,
      • Ricobaraza A.
      • Cuadrado-Tejedor M.
      • Marco S.
      • Pérez-Otaño I.
      • García-Osta A.
      Phenylbutyrate rescues dendritic spine loss associated with memory deficits in a mouse model of Alzheimer disease.
      ).
      In light of the potential clinical utility of HDACi, it has been suggested that HDACi with increased isozyme selectivity and potency would exhibit fewer side effects caused by inhibition of inappropriate isoforms (
      • Allis C.D.
      • Berger S.L.
      • Cote J.
      • Dent S.
      • Jenuwien T.
      • Kouzarides T.
      • Pillus L.
      • Reinberg D.
      • Shi Y.
      • Shiekhattar R.
      • Shilatifard A.
      • Workman J.
      • Zhang Y.
      New nomenclature for chromatin-modifying enzymes.
      ,
      • Arrowsmith C.H.
      • Bountra C.
      • Fish P.V.
      • Lee K.
      • Schapira M.
      Epigenetic protein families: a new frontier for drug discovery.
      ,
      • Subramanian S.
      • Bates S.E.
      • Wright J.J.
      • Espinoza-Delgado I.
      • Piekarz R.L.
      Clinical toxicities of histone deacetylase inhibitors.
      ). Accordingly, significant efforts have been made to identify HDACi with improved HDAC isozyme selectivity (
      • Shakespear M.R.
      • Halili M.A.
      • Irvine K.M.
      • Fairlie D.P.
      • Sweet M.J.
      Histone deacetylases as regulators of inflammation and immunity.
      ,
      • Arrowsmith C.H.
      • Bountra C.
      • Fish P.V.
      • Lee K.
      • Schapira M.
      Epigenetic protein families: a new frontier for drug discovery.
      ,
      • McKinsey T.A.
      Therapeutic potential for HDAC inhibitors in the heart.
      ,
      • Gräff J.
      • Kim D.
      • Dobbin M.M.
      • Tsai L.H.
      Epigenetic regulation of gene expression in physiological and pathological brain processes.
      ,
      • Wiech N.L.
      • Fisher J.F.
      • Helquist P.
      • Wiest O.
      Inhibition of histone deacetylases: A pharmacological approach to the treatment of non-cancer disorders.
      ,
      • Bertrand P.
      Inside HDAC with HDAC inhibitors.
      ,
      • Bradner J.E.
      • West N.
      • Grachan M.L.
      • Greenberg E.F.
      • Haggarty S.J.
      • Warnow T.
      • Mazitschek R.
      Chemical phylogenetics of histone deacetylases.
      ,
      • Chavan A.V.
      • Somani R.R.
      HDAC inhibitors–new generation of target-specific treatment.
      ). Among them, novel selective benzamide-based HDAC1/2 inhibitors have been described that exhibit greater than 100-fold selectivity relative to other HDACs (
      • Baylin S.B.
      • Jones P.A.
      A decade of exploring the cancer epigenome—biological and translational implications.
      ,
      • Methot J.L.
      • Chakravarty P.K.
      • Chenard M.
      • Close J.
      • Cruz J.C.
      • Dahlberg W.K.
      • Fleming J.
      • Hamblett C.L.
      • Hamill J.E.
      • Harrington P.
      • Harsch A.
      • Heidebrecht R.
      • Hughes B.
      • Jung J.
      • Kenific C.M.
      • Kral A.M.
      • Meinke P.T.
      • Middleton R.E.
      • Ozerova N.
      • Sloman D.L.
      • Stanton M.G.
      • Szewczak A.A.
      • Tyagarajan S.
      • Witter D.J.
      • Secrist J.P.
      • Miller T.A.
      Exploration of the internal cavity of histone deacetylase (HDAC) with selective HDAC1/HDAC2 inhibitors (SHI-1:2).
      ).
      Here, we investigated the pharmacological and biological properties of the pan-HDACi SAHA and trichostatin-A (TSA), the class I-selective HDACi MS-275, and two HDAC1/2-selective inhibitors referred to herein as compounds 1 and 2. Using recombinant HDACs, we found that the benzamide HDACi are long residence time inhibitors with slow association and dissociation kinetic rates, whereas the hydroxamates SAHA and TSA possess rapid kinetic binding properties. Crystal structures of SAHA and a representative benzamide compound bound to HDAC2 suggest both chemical and structural reasons for slow binding properties for the benzamides as opposed to the hydroxamate inhibitors. At the cellular level, we discovered that the rate of modulation of histone acetylation by HDACi correlates with the in vitro kinetic properties of the inhibitors, although cell viability and changes in gene expression do not correlate with the inhibitor dissociation rate profiles. This study sheds new light on the functional consequences of using HDACi with different kinetic profiles. In particular, it suggests that the use of HDACi as therapeutic agents should be motivated not only based on their selectivity but also on their kinetic properties.

      DISCUSSION

      In this study, we compared the kinetic binding properties between hydroxamate and benzamide inhibitors and investigated how they affect biological functions in the neuroblastoma cell line SH-SY5Y by examining histone acetylation, cell viability, and gene expression. We confirmed that the hydroxamate inhibitors SAHA and TSA broadly inhibit HDAC isoforms with potency in the submicromolar range, whereas the benzamide inhibitors confer greater HDAC isoform selectivity against only the class I HDACs. MS-275 has submicromolar potency against HDAC1–3, although two other previously described benzamide inhibitors (compound 1 and 2) more potently inhibit only HDAC1 and HDAC2 and do not interfere with the activity of other HDAC isoforms in vitro. The binding affinity of all compounds determined in an orthogonal competitive displacement binding assay were significantly correlated with the inhibitory potencies from the enzymatic assays. Similarity in potency between compound 1, compound 2, and SAHA against HDAC1 and HDAC2 permitted a comparative analysis of the type of zinc chelation moiety used to convey HDAC inhibition, with specific focus on their kinetic binding properties. We employed the Proteros reporter displacement binding assay to quantify the kinetic association and dissociation rates and residence times for each inhibitor against recombinant HDAC1 and HDAC2. The benzamide class of inhibitors consistently displayed slow-on/slow-off binding properties, whereas the hydroxamates exhibited rapid kinetic binding rates. We observed a several orders of magnitude differential in the kinetic rate constants of benzamides compared with hydroxamates, as well as a limitation of this assay platform to quantify extremely rapid kinetic rates in the case of SAHA. Despite chelating zinc in a similar manner to hydroxamic acids, a likely rationale for the slow binding kinetics found with benzamide inhibitors comes from the required disruption of an intramolecular hydrogen bond combined with a protein rearrangement necessary to accommodate the lipophilic biaryl portion of the inhibitor on binding. The binding trajectory either involves traversal of the hydrophobic portion of the ligand past the polar zinc environment or entry through an acetyl release and disposal channel, which contributes to the slow binding kinetics of the benzamides.
      Lysine 5 of histone 2B (H2BK5) and lysine 12 of histone 4 (H4K12) were two marks found in SH-SY5Y cells to exhibit robust increases in acetylation subsequent to HDACi treatment, with SAHA conveying the highest change in histone acetylation compared with MS-275, which is perhaps explained by the difference in potency or selectivity between these two classes of compounds. Increases of histone acetylation at these marks follow the intrinsic inhibitor kinetic properties; hydroxamates provoked a rapid increase of H2BK5 and H4K12 acetylation, whereas benzamides increased histone acetylation more slowly. On cellular washout of these inhibitors, acetylation rapidly decreased to basal levels with the hydroxamates, whereas that for the benzamides was unaffected by compound removal, similar to constant drug exposure. These findings suggest that at the cellular level, the kinetics of acetylation follow the kinetic association and dissociation rate profiles of HDAC inhibitors determined biochemically.
      It has previously been shown that HDAC inhibitors such as SAHA and MS-275 promote cell cycle arrest and apoptosis, linking HDACi to their anti-proliferative and anti-tumor activity (
      • Allis C.D.
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      • Dent S.
      • Jenuwien T.
      • Kouzarides T.
      • Pillus L.
      • Reinberg D.
      • Shi Y.
      • Shiekhattar R.
      • Shilatifard A.
      • Workman J.
      • Zhang Y.
      New nomenclature for chromatin-modifying enzymes.
      ,
      • Baylin S.B.
      • Jones P.A.
      A decade of exploring the cancer epigenome—biological and translational implications.
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      MolProbity: all-atom structure validation for macromolecular crystallography.
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      • Rifkind R.A.
      • Marks P.A.
      Histone deacetylase inhibitor selectively induces p21WAF1 expression and gene-associated histone acetylation.
      ,
      • Rosato R.R.
      • Almenara J.A.
      • Grant S.
      The histone deacetylase inhibitor MS-275 promotes differentiation or apoptosis in human leukemia cells through a process regulated by generation of reactive oxygen species and induction of p21CIP1/WAF1 1.
      ) and to their cellular toxicity (
      • Shakespear M.R.
      • Halili M.A.
      • Irvine K.M.
      • Fairlie D.P.
      • Sweet M.J.
      Histone deacetylases as regulators of inflammation and immunity.
      ,
      • Arrowsmith C.H.
      • Bountra C.
      • Fish P.V.
      • Lee K.
      • Schapira M.
      Epigenetic protein families: a new frontier for drug discovery.
      ,
      • McKinsey T.A.
      Therapeutic potential for HDAC inhibitors in the heart.
      ,
      • Gräff J.
      • Kim D.
      • Dobbin M.M.
      • Tsai L.H.
      Epigenetic regulation of gene expression in physiological and pathological brain processes.
      ,
      • Wiech N.L.
      • Fisher J.F.
      • Helquist P.
      • Wiest O.
      Inhibition of histone deacetylases: A pharmacological approach to the treatment of non-cancer disorders.
      ,
      • Subramanian S.
      • Bates S.E.
      • Wright J.J.
      • Espinoza-Delgado I.
      • Piekarz R.L.
      Clinical toxicities of histone deacetylase inhibitors.
      ,
      • Bradner J.E.
      • West N.
      • Grachan M.L.
      • Greenberg E.F.
      • Haggarty S.J.
      • Warnow T.
      • Mazitschek R.
      Chemical phylogenetics of histone deacetylases.
      ,
      • Khan N.
      • Jeffers M.
      • Kumar S.
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      • Boldog F.
      • Khramtsov N.
      • Qian X.
      • Mills E.
      • Berghs S.C.
      • Carey N.
      • Finn P.W.
      • Collins L.S.
      • Tumber A.
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      Determination of the class and isoform selectivity of small-molecule histone deacetylase inhibitors.
      ,
      • Chou C.J.
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      Pimelic diphenylamide 106 is a slow, tight-binding inhibitor of class I histone deacetylases.
      ). The dramatic decrease in the levels of histone H2B and H4 after 96 h of constant drug exposure in the acetylation studies suggests that cell viability is compromised. Indeed, our results show that constant treatment for 96 h with either hydroxamate or benzamide-containing inhibitors leads to a dramatic induction of cytotoxicity as monitored via detection of cellular ATP. As expected, transient treatment of cells with hydroxamates followed by inhibitor removal reduces the induction of cytotoxicity to near undetectable levels, in line with their acetylation profile and rapid dissociation kinetic profiles. However, it was somewhat surprising to observe that after washout, induction of cytotoxicity from benzamide inhibitors was similarly reduced, despite the maintenance of hyperacetylation up to 48 h. We observed this difference for all inhibitors tested and are the first to report discordance between histone acetylation and cell cytotoxicity. It is likely that pulsed inhibitor treatments limit the activation or repression of specific genes, which may depend exclusively on the onset kinetics of HDACi. Therefore, we speculate that in the case of HDACi with fast dissociation kinetic rates such hydroxamates, drug removal rapidly allows HDACs to return the expression of genes involved in the control of cell cycle and/or cell death to a repressed state, whereas in the case of the benzamides, the duration of the pulse may limit the up-regulation of these genes in the first place, consistent with their slow binding kinetics.
      Despite the differences in the modulation of gene expression between the two classes of HDACi, we also observed a loss of ∼70% gene regulation regardless of the inhibitor used when we compared gene expression profiles of constant and pulsed treatments. This global effect was rather unexpected considering the difference in histone acetylation kinetics after hydroxamate or benzamide treatments, and it implicates a new appreciation for the disconnect between histone acetylation and gene expression when using HDACi. In addition, because we observed a dramatic effect of HDACi on cell viability that seems to depend more on the exposure conditions than inhibitor selectivity, we asked whether the expression of genes involved in the regulation of the cell cycle would be affected by HDACi according to the treatment regimen and not based on their selectivity profile. Previous studies showed that enhanced histone acetylation induced by HDACi allowed the overexpression of genes involved in cell cycle arrest such as cyclin-dependent kinase inhibitors or the growth arrest and DNA damage-inducible protein GADD45 gene family (
      • Baylin S.B.
      • Jones P.A.
      A decade of exploring the cancer epigenome—biological and translational implications.
      ,
      • Methot J.L.
      • Chakravarty P.K.
      • Chenard M.
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      • Dahlberg W.K.
      • Fleming J.
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      • Stanton M.G.
      • Szewczak A.A.
      • Tyagarajan S.
      • Witter D.J.
      • Secrist J.P.
      • Miller T.A.
      Exploration of the internal cavity of histone deacetylase (HDAC) with selective HDAC1/HDAC2 inhibitors (SHI-1:2).
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      Parallel medicinal chemistry approaches to selective HDAC1/HDAC2 inhibitor (SHI-1:2) optimization.
      ,
      • Richon V.M.
      • Sandhoff T.W.
      • Rifkind R.A.
      • Marks P.A.
      Histone deacetylase inhibitor selectively induces p21WAF1 expression and gene-associated histone acetylation.
      ,
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      Induction and superinduction of growth arrest and DNA damage gene 45 (GADD45) α and β messenger RNAs by histone deacetylase inhibitors trichostatin A (TSA) and butyrate in SW620 human colon carcinoma cells.
      ,
      • Rosato R.R.
      • Almenara J.A.
      • Grant S.
      The histone deacetylase inhibitor MS-275 promotes differentiation or apoptosis in human leukemia cells through a process regulated by generation of reactive oxygen species and induction of p21CIP1/WAF1 1.
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      The novel histone deacetylase inhibitor, LBH589, induces expression of DNA damage response genes and apoptosis in Ph− acute lymphoblastic leukemia cells.
      ). Our Gene Ontology analysis revealed that the cyclin-dependent kinase activity pathway was up-regulated by a 24-h constant exposure to SAHA and C1. However, we observed a marked drop in the expression of almost every gene in the pathway when employing a 6-h pulse treatment. Among activated genes that subside with pulsed HDACi administration are genes known to be powerful effectors of cell cycle arrest in multiple cell types and may promote apoptosis (
      • Baylin S.B.
      • Jones P.A.
      A decade of exploring the cancer epigenome—biological and translational implications.
      ,
      • Bolden J.E.
      • Peart M.J.
      • Johnstone R.W.
      Anticancer activities of histone deacetylase inhibitors.
      ). It is possible that prolonged derepression of such genes during constant inhibitor treatment mediates cell cytotoxicity, whereas a 6-h pulse treatment is insufficient to induce an appropriate oncogenic shock capable of initiating cell death. The rapidly reversible expression levels of these genes suggest that an expression threshold must be maintained for greater than 24 h to direct cells toward an apoptotic pathway. This is in agreement with the greatest induction of cell cytotoxicity seen with constant inhibitor treatment for 96 h, or roughly two cell division cycles in this case. As stated previously, most functional gene classes showed an ability to linger in the regulated state past inhibitor removal and as such separate themselves from inhibitor dissociation kinetic rates.
      Qualitatively, we also observed that the changes in histone acetylation level induced by SAHA or TSA seemed to be more pronounced compared with C1 or C2 (Fig. 4). Similarly, gene expression analysis revealed that the total number of genes regulated by SAHA is ∼2.5-fold higher than for C1 after a constant 24-h treatment (Fig. 6). This difference may reflect that the hydroxamates possess a broader HDAC isoform inhibition profile. For example, it has recently been shown that HDAC3 can affect the acetylation of H2B and H4 in vitro (
      • Johnson C.A.
      Human class I histone deacetylase complexes show enhanced catalytic activity in the presence of ATP and co-immunoprecipitate with the ATP-dependent chaperone protein Hsp70.
      ). Moreover, HDAC3 has been suggested to be involved in carcinogenesis by affecting cell differentiation and apoptosis (
      • Wilson A.J.
      Histone deacetylase 3 (HDAC3) and other class I HDACs regulate colon cell maturation and p21 expression and are deregulated in human colon cancer.
      ). Therefore, we cannot exclude the possibility that some of the differences we observed between hydroxamate and benzamide are not only due to their different kinetic properties but due to their different HDAC isoform selectivity profile.
      Apart from modulating histone acetylation, HDACs are also known to deacetylate non-histone proteins, including signaling molecules, structural proteins, chaperon proteins, DNA repair enzymes, transcription factors, and transcription regulators (
      • Kim H.-J.
      • Bae S.C.
      Histone deacetylase inhibitors: molecular mechanisms of action and clinical trials as anti-cancer drugs.
      ,
      • Xu W.S.
      • Parmigiani R.B.
      • Marks P.A.
      Histone deacetylase inhibitors: molecular mechanisms of action.
      ). Interestingly, the level of acetylation of tumor suppressors such as p53, c-Myc, and RUNX3, which is controlled by histone acetyltransferase/HDAC, regulates their function and/or their stability and thus affects their role in cell proliferation, differentiation, and cell death. Therefore, we cannot rule out that non-histone substrates of HDACi and expression-independent events may also underlie this difference in cytotoxicity. Moreover, recent studies have shown that other post-translational modifications such histone methylation, ubiquitination, and phosphorylation are regulated in concert (
      • Vermeulen M.
      • Eberl H.C.
      • Matarese F.
      • Marks H.
      • Denissov S.
      • Butter F.
      • Lee K.K.
      • Olsen J.V.
      • Hyman A.A.
      • Stunnenberg H.G.
      • Mann M.
      Quantitative interaction proteomics and genome-wide profiling of epigenetic histone marks and their readers.
      ,
      • Wang Z.
      • Zang C.
      • Rosenfeld J.A.
      • Schones D.E.
      • Barski A.
      • Cuddapah S.
      • Cui K.
      • Roh T.-Y.
      • Peng W.
      • Zhang M.Q.
      • Zhao K.
      Combinatorial patterns of histone acetylations and methylations in the human genome.
      ,
      • Campos E.I.
      • Reinberg D.
      Histones: annotating chromatin.
      ). We also observed that the methylation of H3K9 was decreased after 24 h of SAHA or MS-275 treatment (Fig. 5), suggesting that the inhibition of HDAC affects the molecular mechanism involved in histone methylation as well. It is thus possible that the difference in gene expression and cytotoxicity that we observe between 6 and 24 h is also due to post-translational histone modification other than acetylation.
      The translation between slow inhibitor binding kinetics and sustained pharmacodynamic modulation has been studied for a number of target classes (
      • Copeland R.A.
      • Pompliano D.L.
      • Meek T.D.
      Drug-target residence time and its implications for lead optimization.
      ). Interpretation of this relationship for the HDAC family is confounded by the downstream epigenetic role of these enzymes to modulate transcriptional activity and by the variety of their non-histone substrates. We have shown discordance between the mechanistic readout of histone acetylation and induction of cell cytotoxicity. We have furthermore demonstrated that global transcriptional modification subsequent to HDAC inhibition more closely correlates with cytotoxicity than with histone acetylation, and we provided evidence that certain gene families might be responsible for alteration of the cell cycle leading to cell death. Our study suggests that the evaluation of HDACi potency in vitro using recombinant proteins and of their pharmacodynamic properties as measured with histone acetylation are insufficient to predict their functional consequences on biological activity such as gene expression and cell viability, and therefore might be misleading in identifying useful HDACi. Although increasing the selective properties of HDACi might be important to prevent the inhibition of inappropriate HDAC isoforms, we propose that a more detailed understanding of how the kinetic properties of inhibition affect cell function will be additionally crucial to select HDACi with suitable pharmacological and biological properties.

      Acknowledgments

      We thank Yvonne Franke and Krista Bowman at Genentech for cloning and expression of HDACs. Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource, a Directorate of SLAC National Accelerator Laboratory and an Office of Science User Facility operated for the United States Department of Energy, Office of Science, by Stanford University. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by National Institutes of Health Grant P41GM103393 from NIGMS and Grant P41RR001209 from the NCRR. Use of the Advanced Photon Source, an Office of Science User Facility operated for the United States Department of Energy Office of Science by Argonne National Laboratory, was supported by the United States Department of Energy under Contract DE-AC02-06CH11357.

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