Richard A. Rifkind

Structures of a histone deacetylase homologue bound to the TSA and SAHA inhibitors.

Histone deacetylases (HDACs) mediate changes in nucleosome conformation and are important in the regulation of gene expression. HDACs are involved in cell-cycle progression and differentiation, and their deregulation is associated with several cancers. HDAC inhibitors, such as trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), have anti-tumour effects, as they can inhibit cell growth, induce terminal differentiation and prevent the formation of tumours in mice models, and they are effective in the treatment of promyelocytic leukemia. Here we describe the structure of the histone deacetylase catalytic core, as revealed by the crystal structure of a homologue from the hyperthermophilic bacterium Aquifex aeolicus, that shares 35.2% identity with human HDAC1 over 375 residues, deacetylates histones in vitro and is inhibited by TSA and SAHA. The deacetylase, deacetylase–TSA and deacetylase–SAHA structures reveal an active site consisting of a tubular pocket, a zinc-binding site and two Asp–His charge-relay systems, and establish the mechanism of HDAC inhibition. The residues that make up the active site and contact the inhibitors are conserved across the HDAC family. These structures also suggest a mechanism for the deacetylation reaction and provide a framework for the further development of HDAC inhibitors as anti-tumour agents.

Histone deacetylase inhibitors as new cancer drugs.

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.

The histone deacetylase inhibitor SAHA arrests cancer cell growth, up-regulates thioredoxin-binding protein-2, and down-regulates thioredoxin

Suberoylanilide hydroxamic acid (SAHA) is a potent inhibitor of histone deacetylases (HDACs) that causes growth arrest, differentiation, and/or apoptosis of many tumor types in vitro and in vivo. SAHA is in clinical trials for the treatment of cancer. HDAC inhibitors induce the expression of less than 2% of genes in cultured cells. In this study we show that SAHA induces the expression of vitamin D-up-regulated protein 1/thioredoxin-binding protein-2 (TBP-2) in transformed cells. As the expression of TBP-2 mRNA is increased, the expression of a second gene, thioredoxin, is decreased. In transient transfection assays, HDAC inhibitors induce TBP-2 promoter constructs, and this induction requires an NF-Y binding site. We report here that TBP-2 expression is reduced in human primary breast and colon tumors compared with adjacent tissue. These results support a model in which the expression of a subset of genes (i.e., including TBP-2) is repressed in transformed cells, leading to a block in differentiation, and culture of transformed cells with SAHA causes re-expression of these genes, leading to induction of growth arrest, differentiation, and/or apoptosis.

Histone deacetylase inhibitors induce remission in transgenic models of therapy-resistant acute promyelocytic leukemia

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.

Cloning and characterization of a histone deacetylase, HDAC9

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.

Suberoylanilide hydroxamic acid (vorinostat) represses androgen receptor expression and acts synergistically with an androgen receptor antagonist to inhibit prostate cancer cell proliferation

Growth of prostate cancer cells is initially dependent on androgens, and androgen ablation therapy is used to control tumor growth. Unfortunately, resistance to androgen ablation therapy inevitably occurs, and there is an urgent need for better treatments for advanced prostate cancer. Histone deacetylase inhibitors, such as suberoylanilide hydroxamic acid (SAHA; vorinostat), are promising agents for the treatment of a range of malignancies, including prostate cancer. SAHA inhibited growth of the androgen-responsive LNCaP prostate cancer cell line at low micromolar concentrations and induced caspase-dependent apoptosis associated with chromatin condensation, DNA fragmentation, and mitochondrial membrane depolarization at higher concentrations (≥5 μmol/L). Gene profiling and immunoblot analyses showed a decrease in androgen receptor (AR) mRNA and protein in LNCaP cells cultured with SAHA compared with control cells, with a corresponding decrease in levels of the AR-regulated gene, prostate-specific antigen. Culture of LNCaP cells in steroid-free medium markedly sensitized the cells to SAHA. Moreover, a combination of low, subeffective doses of SAHA and the AR antagonist bicalutamide resulted in a synergistic reduction in cell proliferation and increase in caspase-dependent cell death. Addition of exogenous androgen prevented the induction of cell death, indicating that suppression of androgen signaling was required for synergy. At the subeffective concentrations, these agents had no effect, alone or in combination, on proliferation or death of AR-negative PC-3 prostate cancer cells. Our findings indicate that SAHA is effective in targeting the AR signaling axis and that androgen deprivation sensitizes prostate cancer cells to SAHA. Consequently, combinatorial treatments that target different components of the AR pathway may afford a more effective strategy to control the growth of prostate cancer cells. [Mol Cancer Ther 2007;6(1):51–60]

Erythroid cell development in fetal mice: Synthetic capacity for different proteins

Abstract Fetal mice of the C57BL/6J strain were used in a study of the rates of synthesis of RNA, total protein and hemoglobin by developing erythroid cells derived from two different sites of erythropoiesis, namely, yolk-sac blood islands and liver. In fetal mice, erythropoiesis proceeds initially in yolk-sac blood islands (8 to 12 days of gestation) and, subsequently, in liver (12 to at least 16 days). In yolk-sac erythroid cells, RNA content and RNA synthesis decrease markedly between the eleventh and thirteenth days of gestation. The rate of synthesis of non-heme protein in these cells declines rapidly from day 12 to 13, while the rate of hemoglobin formation remains essentially unchanged from day 11 through day 13. Actinomycin D inhibits the synthesis of RNA and non-heme protein, but not hemoglobin formation by yolk-sac erythroid cells of the 11-day fetus. In erythroid cells developing in liver, the rate of hemoglobin synthesis, averaged per cell, remains essentially unchanged between day 13 and 15. The rate of hemoglobin synthesis in circulating non-nucleated liver-derived erythroid cells is about threefold that in the population of nucleated erythroid cell precursors in the liver. Actinomycin inhibits hemoglobin synthesis by liver erythroid cells prepared from the liver of the 13-day fetus but not from 14-, 15- or 16-day fetuses. These data suggest that in yolk-sac erythroid cells, non-heme protein formation is limited by a relatively short-lived component essential to protein synthesis, which may be messenger RNA. Hemoglobin formation in yolk-sac erythroid cells after day 11 and in liver erythroid cells after day 14 appears to proceed without a dependence on new RNA synthesis.

Accumulation of α- and β-globin messenger RNAs in mouse erythroleukemia cells

Abstract The accumulation of α- and β-globin mRNA sequences in murine erythroleukemia cells (MELC) treated with various inducers has been studied using specific α- and β-globin complementary DNAs (cDNAs). In cells cultured with dimethylsulfoxide (Me 2 SO), hexamethylene bisacetamide (HMBA) or butyric acid, accumulation of α-globin mRNA is detectable after 16, 12 and 8 hr of culture, respectively. An increase in β-globin mRNA sequences is not detected until 20–24 hr after culture. In cells exposed to hemin, both α- and β-globin mRNAs are detectable by 6 hr of culture, and a constant ratio of αβ-mRNA is maintained during induction. In maximally induced cells, the αβ-globin mRNA ratios are approximately 1 in cells induced by Me 2 SO and HMBA, and 0.66 and 0.3–0.50 in cells induced by butyric acid and hemin, respectively. Thus different inducers of erythroid differentiation in MELC lead to different times of onset of the expression of α- and β-like genes. In addition, the relative accumulation of α- and β-globin mRNAs in induced cells differs with various types of inducers.

Induction of erythroid differentiation by dimethylsulfoxide in cells infected with Friend virus: relationship to the cell cycle.

Cells infected with Friend virus can be induced to erythroid differentiation by culture with 2% dimethylsulfoxide. This study was designed to determine if dimethylsulfoxide causes the expression of erythroid differentiation by an effect on a particular phase of the cell division cycle. The infected cells were synchronized by exposure to 2 mM thymidine. It is shown that dimethylsulfoxide must be present during DNA synthesis (S-phase) and, possibly, shortly thereafter, to induce differentiation assayed by measuring hemoglobin synthesis. In order to achieve an effective intracellular incorporation of dimethylsulfoxide, cells must be exposed to the agent for at least 24-30 hr before the critical S phase. It is suggested that induction of erythroid differentiation in cells infected with Friend virus involves an effect of dimethylsulfoxide, or a metabolic product, that alters the program of transcription, during or immediately after DNA synthesis.

IMMUNOCHEMICAL STAINING FOR ELECTRON MICROSCOPY

The successful al)plication of imntunofluorescent staining in so many branches of biological science has prompted a search for a comparable antibody label which can utilize the higher resolution afforded by electron microscopy. Such a label must have sufficient size and electron-scattering power to be readily discernible against the usual background of biological specimens. A number of methods for employing the specificity of the antibody-antigen reaction for electron microscopic cytology have been proposed and tried. By means of unmodified antibody alone (13) and even more effectively by antibody substituted with heavy metals (1 1 , 12, 22) the presence of antigen may be recognized by virtue of a diffuse increase in electron-scattering due to the bound antibody. Individual antibody molecules and their specific sites of attachment cannot be readily discerned, however, by these techniques. In 1959 Singer proposed the use of the ironcontaining protein, ferritin, a.s a label which would permit the recognition of single antibody molecules (18). Coupling of antibody and fermitin may be accomplished by means of a number of small, bifunctional molecules including nz-xylylene diisocyanate (18), toluene diisocyanate (19) and p ,p’-difiuoro-nu ,m’-dinitrophenylsulfone (14). In each case one active group is available for reaction with ferritin and the other with globulin. The chemical aspects of these reactions and some of the properties of the conjugates have been described (3, 14, 17, 19, 21). Itis the purpose of this communication to elaborate upon certain aspect-s of Singer’s original conjugation procedure which appear to be responsible for a relatively high rate of successful coupling and a satisfactory yield of conjugated antibody. In addition, examples of