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1.
J Cell Biochem ; 107(4): 600-8, 2009 Jul 01.
Article in English | MEDLINE | ID: mdl-19459166

ABSTRACT

The role of histone deacetylases (HDAC) and the potential of these enzymes as therapeutic targets for cancer, neurodegenerative diseases and a number of other disorders is an area of rapidly expanding investigation. There are 18 HDACs in humans. These enzymes are not redundant in function. Eleven of the HDACs are zinc dependent, classified on the basis of homology to yeast HDACs: Class I includes HDACs 1, 2, 3, and 8; Class IIA includes HDACs 4, 5, 7, and 9; Class IIB, HDACs 6 and 10; and Class IV, HDAC 11. Class III HDACs, sirtuins 1-7, have an absolute requirement for NAD(+), are not zinc dependent and generally not inhibited by compounds that inhibit zinc dependent deacetylases. In addition to histones, HDACs have many nonhistone protein substrates which have a role in regulation of gene expression, cell proliferation, cell migration, cell death, and angiogenesis. HDAC inhibitors (HDACi) have been discovered of different chemical structure. HDACi cause accumulation of acetylated forms of proteins which can alter their structure and function. HDACi can induce different phenotypes in various transformed cells, including growth arrest, apoptosis, reactive oxygen species facilitated cell death and mitotic cell death. Normal cells are relatively resistant to HDACi induced cell death. Several HDACi are in various stages of development, including clinical trials as monotherapy and in combination with other anti-cancer drugs and radiation. The first HDACi approved by the FDA for cancer therapy is suberoylanilide hydroxamic acid (SAHA, vorinostat, Zolinza), approved for treatment of cutaneous T-cell lymphoma.


Subject(s)
Enzyme Inhibitors/pharmacology , Histone Deacetylase Inhibitors , Neoplasms/drug therapy , Antineoplastic Agents/pharmacology , Cell Physiological Phenomena/drug effects , Enzyme Inhibitors/classification , Humans , Neoplasms/metabolism , Neoplasms/pathology , Zinc
2.
Proc Natl Acad Sci U S A ; 105(28): 9633-8, 2008 Jul 15.
Article in English | MEDLINE | ID: mdl-18606987

ABSTRACT

Eighteen histone deacetylases (HDACs) are present in humans, categorized into two groups: zinc-dependent enzymes (HDAC1-11) and NAD(+)-dependent enzymes (sirtuins 1-7). Among zinc-dependent HDACs, HDAC6 is unique. It has a cytoplasmic localization, two catalytic sites, a ubiquitin-binding site, and it selectively deacetylases alpha-tubulin and Hsp90. Here, we report the discovery that the redox regulatory proteins, peroxiredoxin (Prx) I and Prx II are specific targets of HDAC6. Prx are antioxidants enzymes whose main function is H(2)O(2) reduction. Prx are elevated in many cancers and neurodegenerative diseases. The acetylated form of Prx accumulates in the absence of an active HDAC6. Acetylation of Prx increases its reducing activity, its resistance to superoxidation, and its resistance to transition to high-molecular-mass complexes. Thus, HDAC6 and Prx are targets for modulating intracellular redox status in therapeutic strategies for disorders as disparate as cancers and neurodegenerative diseases.


Subject(s)
Histone Deacetylases/metabolism , Peroxiredoxins/metabolism , Acetylation , Cell Line, Tumor , Histone Deacetylase 6 , Histone Deacetylases/analysis , Humans , Oxidation-Reduction , Oxidative Stress , Peroxides/metabolism
3.
Oncogene ; 26(37): 5541-52, 2007 Aug 13.
Article in English | MEDLINE | ID: mdl-17694093

ABSTRACT

This review focuses on the mechanisms of action of histone deacetylase (HDAC) inhibitors (HDACi), a group of recently discovered 'targeted' anticancer agents. There are 18 HDACs, which are generally divided into four classes, based on sequence homology to yeast counterparts. Classical HDACi such as the hydroxamic acid-based vorinostat (also known as SAHA and Zolinza) inhibits classes I, II and IV, but not the NAD+-dependent class III enzymes. In clinical trials, vorinostat has activity against hematologic and solid cancers at doses well tolerated by patients. In addition to histones, HDACs have many other protein substrates involved in regulation of gene expression, cell proliferation and cell death. Inhibition of HDACs causes accumulation of acetylated forms of these proteins, altering their function. Thus, HDACs are more properly called 'lysine deacetylases.' HDACi induces different phenotypes in various transformed cells, including growth arrest, activation of the extrinsic and/or intrinsic apoptotic pathways, autophagic cell death, reactive oxygen species (ROS)-induced cell death, mitotic cell death and senescence. In comparison, normal cells are relatively more resistant to HDACi-induced cell death. The plurality of mechanisms of HDACi-induced cell death reflects both the multiple substrates of HDACs and the heterogeneous patterns of molecular alterations present in different cancer cells.


Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm , Enzyme Inhibitors/pharmacology , Histone Deacetylase Inhibitors , Neoplasms/drug therapy , Gene Expression/drug effects , Histone Deacetylases/classification , Histone Deacetylases/metabolism , Humans , Neoplasms/enzymology , Substrate Specificity
4.
Oncogene ; 26(9): 1351-6, 2007 Feb 26.
Article in English | MEDLINE | ID: mdl-17322921

ABSTRACT

The path to the discovery of suberoylanilide hydroxamic acid (SAHA, vorinostat) began over three decades ago with our studies designed to understand why dimethylsulfoxide causes terminal differentiation of the virus-transformed cells, murine erythroleukemia cells. SAHA can cause growth arrest and death of a broad variety of transformed cells both in vitro and in vivo at concentrations that have little or no toxic effects on normal cells. It was discovered that SAHA inhibits the activity of histone deacetylases (HDACs), including all 11 known human class I and class II HDACs. HDACs have many protein targets whose structure and function are altered by acetylation including histones and non-histone proteins component of transcription factors controlling gene expression and proteins that regulate cell proliferation, migration and death. SAHA is in clinical trials and has significant anticancer activity against both hematologic and solid tumors at doses well tolerated by patients. A new drug application has been approved for SAHA (vorinostat) treatment of cutaneous T-cell lymphoma.


Subject(s)
Antineoplastic Agents/chemistry , Enzyme Inhibitors/chemistry , Animals , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Clinical Trials as Topic , Drug Design , Enzyme Inhibitors/therapeutic use , Histone Deacetylase Inhibitors , Humans
5.
Proc Natl Acad Sci U S A ; 102(3): 673-8, 2005 Jan 18.
Article in English | MEDLINE | ID: mdl-15637150

ABSTRACT

This study examines the basis of resistance and sensitivity of normal and transformed cells to histone deacetylase inhibitor (HDACi)-induced cell death, specifically the role of caspases and thioredoxin (Trx). An important attribute of HDACis is that they induce cancer cell death at concentrations to which normal cells are relatively resistant, making them well suited for cancer therapy. The mechanism underlying this selectivity has not been understood. In this study we found that the HDACi suberoylanilide hydroxamic acid (SAHA) and MS-275, a benzamide, cause an accumulation of reactive oxygen species (ROS) and caspase activation in transformed but not normal cells. Inhibition of caspases does not block HDACi-induced cell death. These studies provide a possible mechanism that can explain why normal but not certain transformed cells are resistant to HDACi-induced cell death. The HDACi causes an increase in the level of Trx, a major reducing protein for many targets, in normal cells but not in transformed cells. The SAHA-induced increase in Trx activity in normal cells is associated with no increase in ROS accumulation. Transfection of transformed cells with Trx small interfering RNA caused a marked decrease in the level of Trx protein with an increase in ROS, a decrease in cell proliferation, and an increase in sensitivity to SAHA-induced cell death. Thus, Trx, independent of the caspase apoptotic pathway, is an important determinant of resistance of cells to HDACi-induced cell death.


Subject(s)
Histone Deacetylase Inhibitors , Neoplasms/pathology , Thioredoxins , Apoptosis/drug effects , Benzamides/pharmacology , Caspases/metabolism , Cell Line, Transformed , Drug Resistance, Neoplasm , Enzyme Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , Neoplasms/drug therapy , Pyridines/pharmacology , RNA, Small Interfering/pharmacology , Reactive Oxygen Species/metabolism , Thioredoxins/genetics , Vorinostat
6.
Proc Natl Acad Sci U S A ; 101(5): 1241-6, 2004 Feb 03.
Article in English | MEDLINE | ID: mdl-14734806

ABSTRACT

Histone deacetylase (HDAC) inhibitors (HDACi) cause cancer cell growth arrest and/or apoptosis in vivo and in vitro. The HDACi suberoylanilide hydroxamic acid (SAHA) is in phase I/II clinical trials showing significant anticancer activity. Despite wide distribution of HDACs in chromatin, SAHA alters the expression of few genes in transformed cells. p21(WAF1) is one of the most commonly induced. SAHA does not alter the expression of p27(KIPI), an actively transcribed gene, or globin, a silent gene, in ARP-1 cells. Here we studied SAHA-induced changes in the p21(WAF1) promoter of ARP-1 cells to better understand the mechanism of HDACi gene activation. Within 1 h, SAHA caused modifications in acetylation and methylation of core histones and increased DNase I sensitivity and restriction enzyme accessibility in the p21(WAF1) promoter. These changes did not occur in the p27(KIPI) or epsilon-globin gene-related histones. The HDACi caused a marked decrease in HDAC1 and Myc and an increase in RNA polymerase II in proteins bound to the p21(WAF1) promoter. Thus, this study identifies effects of SAHA on p21(WAF1)-associated proteins that explain, at least in part, the selective effect of HDACi in altering gene expression.


Subject(s)
Cyclins/genetics , Enzyme Inhibitors/pharmacology , Histone Deacetylase Inhibitors , Histone Deacetylases/analysis , Hydroxamic Acids/pharmacology , Promoter Regions, Genetic , Acetylation , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21 , Deoxyribonuclease I/pharmacology , Histone Deacetylase 1 , Humans , Protein Processing, Post-Translational , Vorinostat
7.
Cancer Res ; 61(23): 8492-7, 2001 Dec 01.
Article in English | MEDLINE | ID: mdl-11731433

ABSTRACT

Histone deacetylase (HDACs) regulate histone acetylation by catalyzing the removal of acetyl groups on the NH(2)-terminal lysine residues of the core nucleosomal histones. Modulation of the acetylation status of core histones is involved in the regulation of the transcriptional activity of certain genes. HDAC activity is generally associated with transcriptional repression. Aberrant recruitment of HDAC activity has been associated with the development of certain human cancers. We have developed a class of HDAC inhibitors, such as suberoylanilide hydroxamic acid (SAHA), that were initially identified based on their ability to induce differentiation of cultured murine erythroleukemia cells. Additional studies have demonstrated that SAHA inhibits the growth of tumors in rodents. In this study we have examined the effects of SAHA on MCF-7 human breast cancer cells. We found that SAHA causes the inhibition of proliferation, accumulation of cells in a dose-dependent manner in G(1) then G(2)-M phase of the cell cycle, and induction of milk fat globule protein, milk fat membrane globule protein, and lipid droplets. Growth inhibition was associated with morphological changes including the flattening and enlargement of the cytoplasm, and a decrease in the nuclear:cytoplasmic ratio. Withdrawal of SAHA led to reentry of cells into the cell cycle and reversal to a less differentiated phenotype. SAHA induced differentiation in the estrogen receptor-negative cell line SKBr-3 and the retinoblastoma-negative cell line MDA-468. We propose that SAHA has profound antiproliferative activity by causing these cells to undergo cell cycle arrest and differentiation that is dependent on the presence of SAHA. SAHA and other HDAC inhibitors are currently in Phase I clinical trials. These findings may impact the clinical use of these drugs.


Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Enzyme Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , Breast Neoplasms/metabolism , Cell Differentiation/drug effects , Cell Division/drug effects , Cell Line, Transformed , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial Cells/pathology , Histone Deacetylase Inhibitors , Humans , Lipids/biosynthesis , Milk Proteins/biosynthesis , Receptor, ErbB-2/biosynthesis , Receptor, ErbB-2/physiology , Receptors, Estrogen/biosynthesis , Receptors, Estrogen/physiology , Retinoblastoma Protein/biosynthesis , Retinoblastoma Protein/physiology , Vorinostat
8.
J Clin Invest ; 108(9): 1321-30, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11696577

ABSTRACT

Acute promyelocytic leukemia (APL) is associated with chromosomal translocations, invariably involving the retinoic acid receptor alpha (RAR alpha) gene fused to one of several distinct loci, including the PML or PLZF genes, involved in t(15;17) or t(11;17), respectively. Patients with t(15;17) APL respond well to retinoic acid (RA) and other treatments, whereas those with t(11;17) APL do not. The PML-RAR alpha and PLZF-RAR alpha fusion oncoproteins function as aberrant transcriptional repressors, in part by recruiting nuclear receptor-transcriptional corepressors and histone deacetylases (HDACs). Transgenic mice harboring the RAR alpha fusion genes develop forms of leukemia that faithfully recapitulate both the clinical features and the response to RA observed in humans with the corresponding translocations. Here, we investigated the effects of HDAC inhibitors (HDACIs) in vitro and in these animal models. In cells from PLZF-RAR alpha/RAR alpha-PLZF transgenic mice and cells harboring t(15;17), HDACIs induced apoptosis and dramatic growth inhibition, effects that could be potentiated by RA. HDACIs also increased RA-induced differentiation. HDACIs, but not RA, induced accumulation of acetylated histones. Using microarray analysis, we identified genes induced by RA, HDACIs, or both together. In combination with RA, all HDACIs tested overcame the transcriptional repression exerted by the RAR alpha fusion oncoproteins. In vivo, HDACIs induced accumulation of acetylated histones in target organs. Strikingly, this combination of agents induced leukemia remission and prolonged survival, without apparent toxic side effects.


Subject(s)
Enzyme Inhibitors/pharmacology , Histone Deacetylase Inhibitors , Leukemia, Promyelocytic, Acute/drug therapy , Leukemia, Promyelocytic, Acute/genetics , Remission Induction , Animals , Antineoplastic Agents/pharmacology , Apoptosis , Blotting, Northern , Blotting, Western , Cell Cycle , Cell Differentiation , Cell Division , DNA, Complementary/metabolism , Humans , Hydroxamic Acids/pharmacology , In Situ Nick-End Labeling , Mice , Mice, Transgenic , Microscopy, Fluorescence , Models, Chemical , Oligonucleotide Array Sequence Analysis , Phenylbutyrates/pharmacology , Protein Binding , Receptors, Retinoic Acid/genetics , Retinoic Acid Receptor alpha , Time Factors , Transcription, Genetic , Transcriptional Activation , Tumor Cells, Cultured , Up-Regulation , Vorinostat
9.
Curr Opin Oncol ; 13(6): 477-83, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11673688

ABSTRACT

Histone deacetylase inhibitors are potent inducers of growth arrest, differentiation, or apoptotic cell death in a variety of transformed cells in culture and in tumor bearing animals. Histone deacetylases and the family of histone acetyl transferases are involved in determining the acetylation of histones, which play a role in regulation of gene expression. Radiograph crystallographic studies reveal that the histone deacetylase inhibitors, suberoylanilide hydroxamic acid and trichostatin A, fit into the catalytic site of histone deacetylase, which has a tubular structure with a zinc atom at its base. The hydroxamic acid moiety of the inhibitor binds to the zinc. Histone deacetylase inhibitors cause acetylated histones to accumulate in both tumor and peripheral circulating mononuclear cells. Accumulation of acetylated histones has been used as a marker of the biologic activity of the agents. Hydroxamic acid-based histone deacetylase inhibitors limit tumor cell growth in animals with little or no toxicity. These compounds act selectively on genes, altering the transcription of only approximately 2% of expressed genes in cultured tumor cells. A number of proteins other than histones are substrates for histone deacetylases. The role that these other targets play in histone deacetylase inducement of cell growth arrest, differentiation, or apoptotic cell death is not known. This review summarizes the characteristics of a variety of inhibitors of histone deacetylases and their effects on transformed cells in culture and tumor growth in animal models. Several structurally different histone deacetylase inhibitors are in phase I or II clinical trials in patients with cancers.


Subject(s)
Apoptosis/drug effects , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Neoplastic , Histone Deacetylase Inhibitors , Histone Deacetylases/metabolism , Histones/metabolism , Animals , Biomarkers, Tumor , Cell Cycle/drug effects , Cell Differentiation/drug effects , Disease Models, Animal , Humans , Neoplasms/drug therapy , Tumor Cells, Cultured
10.
Proc Natl Acad Sci U S A ; 98(19): 10572-7, 2001 Sep 11.
Article in English | MEDLINE | ID: mdl-11535832

ABSTRACT

Histone deacetylase (HDAC) catalyzes the removal of the acetyl group from the lysine residues in the N-terminal tails of nucleosomal core histones. Eight human HDACs have been identified so far. Here, we report the identification of a ninth member of the HDAC family, designated HDAC9. HDAC9 is a class II HDAC and its gene resides on human chromosome 7. HDAC9 has several alternatively spliced isoforms. One of these isoforms is histone deacetylase-related protein or myocyte enhancer-binding factor 2-interacting transcriptional repressor that we and others have previously reported and which does not possess an HDAC catalytic domain. The longest of the HDAC9 isoforms contains 1,011 aa. The isoform, designated HDAC9a, is 132 aa shorter at the C terminus than HDAC9. Also, we have identified isoforms of HDAC9 that lack the nuclear localization signal. Similar to histone deacetylase-related protein, HDAC9 transcripts are expressed at high levels in brain and skeletal muscle. The ratio of HDAC9 and HDAC9a transcripts differs among the tissues examined. HDAC9 and HDAC9a contain the HDAC catalytic domain, and Flag-tagged HDAC9 and HDAC9a possess deacetylase activity. HDAC9 and HDAC9a also repress myocyte enhancer-binding factor 2-mediated transcription. In the present study, we have identified HDAC9 and a number of alternatively spliced isoforms of HDAC9 with potentially different biological activities.


Subject(s)
Alternative Splicing , Histone Deacetylases/genetics , Repressor Proteins/genetics , Amino Acid Sequence , Base Sequence , Cell Line, Transformed , Cloning, Molecular , DNA, Complementary , DNA-Binding Proteins/metabolism , Gene Expression , Genes, Reporter , Histone Deacetylases/metabolism , Humans , Luciferases/genetics , MEF2 Transcription Factors , Molecular Sequence Data , Myogenic Regulatory Factors , RNA, Messenger , Repressor Proteins/metabolism , Tissue Distribution , Transcription Factors/metabolism , Transcription, Genetic
13.
Clin Cancer Res ; 7(4): 962-70, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11309347

ABSTRACT

PURPOSE: We have synthesized a series of hybrid polar compounds that induce differentiation and/or apoptosis of various transformed cells. These agents are also potent inhibitors of histone deacetylases (HDACs). Pyroxamide (suberoyl-3-aminopyridineamide hydroxamic acid) is a new member of this class of compounds that is currently under development as an anticancer agent. We investigated the activity of pyroxamide as an inducer of differentiation and/or apoptosis in transformed cells. EXPERIMENTAL DESIGN AND RESULTS: Pyroxamide, at micromolar concentrations, induced terminal differentiation in murine erythroleukemia (MEL) cells and caused growth inhibition by cell cycle arrest and/or apoptosis in MEL, prostate carcinoma, bladder carcinoma, and neuroblastoma cells. Administration of pyroxamide (100 or 200 mg/kg/day) to nude mice at doses that caused little evident toxicity significantly suppressed the growth of s.c. CWR22 prostate cancer xenografts. Despite the potent growth-inhibitory effects of pyroxamide in this tumor model, serum prostate-specific antigen levels in control versus pyroxamide-treated mice were not significantly different. Pyroxamide is a potent inhibitor of affinity-purified HDAC1 (ID(50) = 100 nM) and causes the accumulation of acetylated core histones in MEL cells cultured with the agent. Human CWR22 prostate tumor xenografts from mice treated with pyroxamide (100 or 200 mg/kg/day) showed increased levels of histone acetylation and increased expression of the cell cycle regulator p21/WAF1, compared with tumors from vehicle-treated control animals. CONCLUSIONS: The findings suggest that pyroxamide may be a useful agent for the treatment of malignancy and that induction of p21/WAF1 in transformed cells by pyroxamide may contribute to the antitumor effects of this agent.


Subject(s)
Aminopyridines/pharmacology , Antineoplastic Agents/pharmacology , Cell Differentiation/drug effects , Enzyme Inhibitors/pharmacology , Histone Deacetylase Inhibitors , Hydroxamic Acids/pharmacology , Acetylation/drug effects , Aminopyridines/therapeutic use , Animals , Antineoplastic Agents/therapeutic use , Cell Division/drug effects , Cell Line, Transformed , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/biosynthesis , Disease Models, Animal , Enzyme Inhibitors/therapeutic use , Histone Deacetylases/metabolism , Histones/metabolism , Humans , Hydroxamic Acids/therapeutic use , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Prostate-Specific Antigen/blood , Prostatic Neoplasms/blood , Prostatic Neoplasms/drug therapy , Treatment Outcome , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
14.
Cancer Res ; 61(9): 3591-4, 2001 May 01.
Article in English | MEDLINE | ID: mdl-11325825

ABSTRACT

Histone deacetylase inhibitors (HDACIs) inhibit the growth of a variety of transformed cells in culture. We demonstrated previously that the hybrid-polar HDACI m-carboxycinnamic acid bis-hydroxamide (CBHA) induces apoptosis of human neuroblastoma in vitro and is effective in lower doses when combined with retinoids. The current study investigates the effect of CBHA on the growth of human neuroblastoma in vivo, both alone and in combination with all-trans retinoic acid (atRA), using a severe combined immunodeficiency-mouse xenograft model. CBHA (50, 100, and 200 mg/kg/day) inhibited growth of SMS-KCN-69n tumor xenografts in a dose-dependent fashion, with 200 mg/kg CBHA resulting in a complete suppression of tumor growth. The efficacy of 50 and 100 mg/kg CBHA was enhanced by the addition of 2.5 mg/kg atRA. This dose of atRA was ineffective when administered alone. Treatment was accompanied by mild weight loss in all groups except the lowest dose of CBHA. Our results suggest HDACIs alone or combined with retinoids may have therapeutic utility for neuroblastoma.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Cinnamates/pharmacology , Enzyme Inhibitors/pharmacology , Neuroblastoma/drug therapy , Tretinoin/pharmacology , Acetylation , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/toxicity , Antineoplastic Combined Chemotherapy Protocols/toxicity , Cell Division/drug effects , Cinnamates/administration & dosage , Cinnamates/toxicity , Dose-Response Relationship, Drug , Drug Synergism , Enzyme Inhibitors/toxicity , Female , Growth Inhibitors/pharmacology , Histone Deacetylase Inhibitors , Histones/metabolism , Humans , Mice , Mice, SCID , Neuroblastoma/enzymology , Neuroblastoma/pathology , Tretinoin/administration & dosage , Tumor Cells, Cultured , Weight Loss/drug effects , Xenograft Model Antitumor Assays
15.
Proc Natl Acad Sci U S A ; 97(26): 14329-33, 2000 Dec 19.
Article in English | MEDLINE | ID: mdl-11114188

ABSTRACT

Histone deacetylase 4 (HDAC4) is a member of a family of enzymes that catalyze the removal of acetyl groups from core histones, resulting in a compact chromatin structure that is generally associated with repressed gene transcription. Protein phosphorylation has been implicated in the regulation of the corepressor activity of the deacetylase. Here we report that serine/threonine kinases are found in association with HDAC4 and phosphorylate HDAC4 in vitro, and HDAC4 is phosphorylated in cells. The extracellular signal-regulated kinases 1 and 2 (ERK1/2), also known as p44(MAPK) and p42(MAPK), respectively, are two of the kinases associated with HDAC4. ERK1/2 are components of the Ras-mitogen-activated protein kinase (MAPK) signal transduction pathway. Activation of the Ras-MAPK pathway by expression of oncogenic Ras or constitutively active MAPK/ERK kinase 1 results in an increased percentage of cells (from approximately 10% to approximately 70%) that express HDAC4 in the nucleus in C2C12 myoblast cells. In cells transfected with oncogenic Ras, nuclear HDAC4 is associated with kinase activity. Our results provide evidence that protein kinase activity is present in a protein complex with HDAC4 and directly links the Ras-MAPK signal transduction pathway to a mechanism for chromatin remodeling (i.e., histone deacetylation).


Subject(s)
Histone Deacetylases/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinases/metabolism , Oncogene Protein p21(ras)/metabolism , Protein Serine-Threonine Kinases/metabolism , Repressor Proteins/metabolism , Animals , Cell Line , Cell Line, Transformed , Cell Nucleus/metabolism , Cytoplasm/metabolism , Enzyme Activation , Histone Deacetylases/genetics , Humans , MAP Kinase Kinase 1 , Mice , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinase Kinases/metabolism , Phosphorylation , Repressor Proteins/genetics
16.
Med Pediatr Oncol ; 35(6): 577-81, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11107121

ABSTRACT

BACKGROUND: Neuroblastoma is a common childhood cancer with a poor overall prognosis. Retinoic acids (RAs) have been studied as a potential therapy, showing promise in recurrent disease. The histone deacetylase inhibitor (HDACI) M-carboxycinnamic acid bishydroxamide (CBHA) is another potential therapy, which we recently described. Combinations of RAs and HDACIs currently under investigation display synergy in certain neoplasms. In this study, we evaluate the effect of combinations of RAs and HDACIs on human neuroblastoma cells. PROCEDURE: Established cell lines were cultured in increasing concentrations of HDACIs, RAs, and combinations thereof. Following exposure, viable cell number was quantified by trypan blue dye exclusion on a hemacytometer. Cell cycle analysis was performed by propidium iodide staining and FACS. RESULTS: All assayed HDACIs and RAs decreased viable cell number. Lower concentrations of each agent were effective when the two were combined. The primary reason for decreased cell number appears to be apoptosis following HDACI exposure and G1 arrest following RA exposure. Both effects are seen with cotreatment. Caspase inhibition abrogates the apoptotic response. CONCLUSIONS: CBHA causes apoptosis of human neuroblastoma in vitro, an effect that can add to the effects of RA. HDACIs and RAs inhibit neuroblastoma in significantly lower concentrations when used together than when used individually. Combination therapy may improve the ultimate efficacy while reducing the side effects of these agents in clinical use.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cinnamates/therapeutic use , Histone Deacetylase Inhibitors , Neuroblastoma/drug therapy , Neuroblastoma/pathology , Tretinoin/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Caspase Inhibitors , Cell Division/drug effects , Cinnamates/pharmacology , G1 Phase/drug effects , Humans , Tretinoin/pharmacology , Tumor Cells, Cultured
17.
Cancer Res ; 60(18): 5165-70, 2000 Sep 15.
Article in English | MEDLINE | ID: mdl-11016644

ABSTRACT

Suberoylanilide hydroxamic acid (SAHA) is the prototype of a family of hybrid polar compounds that induce growth arrest in transformed cells and show promise for the treatment of cancer. SAHA induces differentiation and/or apoptosis in certain transformed cells in culture and is a potent inhibitor of histone deacetylases. In this study, we examined the effects of SAHA on the growth of human prostate cancer cells in culture and on the growth of the CWR22 human prostate xenograft in nude mice. SAHA suppressed the growth of the LNCaP, PC-3, and TSU-Pr1 cell lines at micromolar concentrations (2.5-7.5 microM). SAHA induced dose-dependent cell death in the LNCaP cells. In mice with transplanted CWR222 human prostate tumors, SAHA (25, 50, and 100 mg/kg/day) caused significant suppression of tumor growth compared with mice receiving vehicle alone; treatment with 50 mg/kg/day resulted in a 97% reduction in the mean final tumor volume compared with controls. At this dose, there was no detectable toxicity as evaluated by weight gain and necropsy examination. Increased accumulation of acetylated core histones was detected in the CWR22 tumors within 6 h of SAHA administration. SAHA induced prostate-specific antigen mRNA expression in CWR22 prostate cancer cells, resulting in higher levels of serum prostate-specific antigen than predicted from tumor volume alone. The results suggest that hydroxamic acid-based hybrid polar compounds inhibit prostate cancer cell growth and may be useful, relatively nontoxic agents for the treatment of prostate carcinoma.


Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , Prostatic Neoplasms/drug therapy , Animals , Antineoplastic Agents/toxicity , Cell Death/drug effects , Cell Division/drug effects , Enzyme Inhibitors/toxicity , Growth Inhibitors/pharmacology , Histone Deacetylase Inhibitors , Histones/metabolism , Humans , Hydroxamic Acids/toxicity , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Transplantation , Prostate-Specific Antigen/blood , Prostatic Neoplasms/enzymology , Prostatic Neoplasms/pathology , Transplantation, Heterologous , Tumor Cells, Cultured/drug effects , Vorinostat
18.
Proc Natl Acad Sci U S A ; 97(18): 10014-9, 2000 Aug 29.
Article in English | MEDLINE | ID: mdl-10954755

ABSTRACT

Histone deacetylases (HDACs) catalyze the removal of acetyl groups on the amino-terminal lysine residues of core nucleosomal histones. This activity is associated generally with transcriptional repression. We have reported previously that inhibition of HDAC activity by hydroxamic acid-based hybrid polar compounds, such as suberoylanilide hydroxamic acid (SAHA), induces differentiation and/or apoptosis of transformed cells in vitro and inhibits tumor growth in vivo. SAHA is a potentially new therapeutic approach to cancer treatment and is in Phase I clinical trials. In several tumor cell lines examined, HDAC inhibitors alter the expression of less than 1% of expressed genes, including the cell cycle kinase inhibitor p21(WAF1). In T24 bladder carcinoma cells, SAHA induces up to a 9-fold increase in p21(WAF1) mRNA and protein, which is, at least in part, because of an increase in the rate of transcription of the gene. SAHA causes an accumulation of acetylated histones H3 and H4 in total cellular chromatin by 2 h, which is maintained through 24 h of culture. An increase in the accumulation of acetylated H3 and H4 was detected throughout the p21(WAF1) promoter and the structural gene after culture with SAHA. The level of histone acetylation did not change in chromatin associated with the actin and p27 genes, and their mRNA expression was not altered during culture of T24 cells with SAHA. Thus, the present findings indicate that the induction of p21(WAF1) by SAHA is regulated, at least in part, by the degree of acetylation of the gene-associated histones and that this induced increase in acetylation is gene selective.


Subject(s)
Anticarcinogenic Agents/pharmacology , Cyclins/genetics , Gene Expression Regulation, Neoplastic , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Hydroxamic Acids/pharmacology , Transcription, Genetic , Cell Nucleus/metabolism , Chromatin/metabolism , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/metabolism , DNA Primers , Enzyme Inhibitors/metabolism , Gene Expression Regulation, Enzymologic , Histone Deacetylase Inhibitors , Histones/metabolism , Humans , Polymerase Chain Reaction , Tumor Cells, Cultured , Urinary Bladder Neoplasms , Vorinostat
19.
J Natl Cancer Inst ; 92(15): 1210-6, 2000 Aug 02.
Article in English | MEDLINE | ID: mdl-10922406

ABSTRACT

Histone deacetylase (HDAC) inhibitors have been shown to be potent inducers of growth arrest, differentiation, and/or apoptotic cell death of transformed cells in vitro and in vivo. One class of HDAC inhibitors, hydroxamic acid-based hybrid polar compounds (HPCs), induce differentiation at micromolar or lower concentrations. Studies (x-ray crystallographic) showed that the catalytic site of HDAC has a tubular structure with a zinc atom at its base and that these HDAC inhibitors, such as suberoylanilide hydroxamic acid and trichostatin A, fit into this structure with the hydroxamic moiety of the inhibitor binding to the zinc. HDAC inhibitors cause acetylated histones to accumulate in both tumor and normal tissues, and this accumulation can be used as a marker of the biologic activity of the HDAC inhibitors. Hydroxamic acid-based HPCs act selectively to inhibit tumor cell growth at levels that have little or no toxicity for normal cells. These compounds also act selectively on gene expression, altering the expression of only about 2% of the genes expressed in cultured tumor cells. In general, chromatin fractions enriched in actively transcribed genes are also enriched in highly acetylated core histones, whereas silent genes are associated with nucleosomes with a low level of acetylation. However, HDACs can also acetylate proteins other than histones in nucleosomes. The role that these other targets play in the induction of cell growth arrest, differentiation, and/or apoptotic cell death has not been determined. Our working hypothesis is that inhibition of HDAC activity leads to the modulation of expression of a specific set of genes that, in turn, result in growth arrest, differentiation, and/or apoptotic cell death. The hydroxamic acid-based HPCs are potentially effective agents for cancer therapy and, possibly, cancer chemoprevention.


Subject(s)
Acetyltransferases/metabolism , Apoptosis/drug effects , Cell Transformation, Neoplastic/drug effects , Enzyme Inhibitors/pharmacology , Histone Deacetylase Inhibitors , Neoplasms/drug therapy , Saccharomyces cerevisiae Proteins , Animals , Gene Expression Regulation, Neoplastic/drug effects , Histone Acetyltransferases , Humans , Neoplasms/enzymology
20.
Proc Natl Acad Sci U S A ; 97(3): 1056-61, 2000 Feb 01.
Article in English | MEDLINE | ID: mdl-10655483

ABSTRACT

Histone deacetylases (HDACs) are involved in regulating transcription by modifying the core histones of the nucleosome. To date, six HDACs have been identified in mammalian cells: the yeast RPD3 homologs HDAC1, 2, and 3 and the yeast HDA1 homologs HDAC4, 5, and 6. HDAC4 and HDAC5 contain a noncatalytic N-terminal domain. Herein, we report the identification of a protein HDRP (HDAC-related protein) that shares 50% identity in deduced amino acid sequence to the noncatalytic N-terminal domain of HDAC4 and 5. The steady-state levels of HDRP mRNA are high in human brain, heart, and skeletal muscle and low in the several other tissues. HDRP has an apparent molecular mass of approximately 75 kDa. HDRP does not possess intrinsic HDAC activity but forms complexes with both HDAC1 and HDAC3. HDRP represses both basal and activated transcription in transient transfection assays when tethered to DNA as a Gal4-fusion protein. HDAC inhibitors do not reverse transcriptional repression mediated by Gal4-HDRP. Thus, HDRP is a transcriptional repressor and can repress transcription in the presence of HDAC inhibitors.


Subject(s)
Histone Deacetylases/chemistry , Repressor Proteins/chemistry , Repressor Proteins/isolation & purification , Amino Acid Sequence , Animals , COS Cells , Chlorocebus aethiops , Histone Deacetylase 1 , Histone Deacetylases/metabolism , Humans , Molecular Sequence Data , Organ Specificity , RNA, Messenger/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Repressor Proteins/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Transcription, Genetic , Transfection
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