Crystal structure of the BTB domain from PLZF

The BTB domain (also known as the POZ domain) is an evolutionarily conserved protein–protein interaction motif found at the N terminus of 5–10% of C2H2-type zinc-finger transcription factors, as well as in some actin-associated proteins bearing the kelch motif. Many BTB proteins are transcriptional regulators that mediate gene expression through the control of chromatin conformation. In the human promyelocytic leukemia zinc finger (PLZF) protein, the BTB domain has transcriptional repression activity, directs the protein to a nuclear punctate pattern, and interacts with components of the histone deacetylase complex. The association of the PLZF BTB domain with the histone deacetylase complex provides a mechanism of linking the transcription factor with enzymatic activities that regulate chromatin conformation. The crystal structure of the BTB domain of PLZF was determined at 1.9 Å resolution and reveals a tightly intertwined dimer with an extensive hydrophobic interface. Approximately one-quarter of the monomer surface area is involved in the dimer intermolecular contact. These features are typical of obligate homodimers, and we expect the full-length PLZF protein to exist as a branched transcription factor with two C-terminal DNA-binding regions. A surface-exposed groove lined with conserved amino acids is formed at the dimer interface, suggestive of a peptide-binding site. This groove may represent the site of interaction of the PLZF BTB domain with nuclear corepressors or other nuclear proteins.

Macrophage inflammatory protein-2: Chromosomal regulation in rat small intestinal epithelial cells

Nonpathogenic, resident bacteria participate in the pathogenesis of inflammation in the small intestine, but the molecular messages produced by such bacteria are unknown. Inflammatory responses involve the recruitment of specific leukocyte subsets. We, therefore, hypothesized that butyrate, a normal bacterial metabolite, may modulate chemokine secretion by epithelial cells, by amplifying their response to proinflammatory signals. We studied the expression of the chemokine, macrophage inflammatory protein-2 (MIP-2) by the rat small intestinal epithelial cell line, IEC-6. Cells were stimulated with lipopolysaccharide or with interleukin 1β (IL-1β) and incubated with sodium butyrate. Acetylation of histones was examined in Triton X acetic acid–urea gels by PAGE. Unstimulated IEC-6 cells did not secrete MIP-2. However, lipopolysaccharide and IL-1β induced MIP-2 expression. Butyrate enhanced MIP-2 secretion both in lipopolysaccharide-stimulated and IL-1β-stimulated enterocytes; but butyrate alone did not induce MIP-2 expression. Butyrate increased the acetylation of histones extracted from the nuclei of IEC-6 cells. Furthermore, acetylation of histones (induced by trichostatin A, a specific inhibitor of histone deacetylase) enhanced MIP-2 expression by cells stimulated with IL-1β. In conclusion, trichostatin A reproduced the effects of butyrate on MIP-2 secretion. Butyrate, therefore, increases MIP-2 secretion in stimulated cells by increasing histone acetylation. We speculate that butyrate carries information from bacteria to epithelial cells. Epithelial cells transduce this signal through histone deacetylase, modulating the secretion of chemokines.

Corepressor SMRT binds the BTB/POZ repressing domain of the LAZ3/BCL6 oncoprotein

The LAZ3/BCL6 (lymphoma-associated zinc finger 3/B cell lymphomas 6) gene frequently is altered in non-Hodgkin lymphomas. It encodes a sequence-specific DNA binding transcriptional repressor that contains a conserved N-terminal domain, termed BTB/POZ (bric-à-brac tramtrack broad complex/pox viruses and zinc fingers). Using a yeast two-hybrid screen, we show here that the LAZ3/BCL6 BTB/POZ domain interacts with the SMRT (silencing mediator of retinoid and thyroid receptor) protein. SMRT originally was identified as a corepressor of unliganded retinoic acid and thyroid receptors and forms a repressive complex with a mammalian homolog of the yeast transcriptional repressor SIN3 and the HDAC-1 histone deacetylase. Protein binding assays demonstrate that the LAZ3/BCL6 BTB/POZ domain directly interacts with SMRT in vitro. Furthermore, DNA-bound LAZ3/BCL6 recruits SMRT in vivo, and both overexpressed proteins completely colocalize in nuclear dots. Finally, overexpression of SMRT enhances the LAZ3/BCL6-mediated repression. These results define SMRT as a corepressor of LAZ3/BCL6 and suggest that LAZ3/BCL6 and nuclear hormone receptors repress transcription through shared mechanisms involving SMRT recruitment and histone deacetylation.

Targeted chromatin binding and histone acetylation in vivo by thyroid hormone receptor during amphibian development

Amphibian metamorphosis is marked by dramatic, thyroid hormone (TH)-induced changes involving gene regulation by TH receptor (TR). It has been postulated that TR-mediated gene regulation involves chromatin remodeling. In the absence of ligand, TR can repress gene expression by recruiting a histone deacetylase complex, whereas liganded TR recruits a histone acetylase complex for gene activation. Earlier studies have led us to propose a dual function model for TR during development. In premetamorphic tadpoles, unliganded TR represses transcription involving histone deacetylation. During metamorphosis, endogenous TH allows TR to activate gene expression through histone acetylation. Here using chromatin immunoprecipitation assay, we directly demonstrate TR binding to TH response genes constitutively in vivo in premetamorphic tadpoles. We further show that TH treatment leads to histone deacetylase release from TH response gene promoters. Interestingly, in whole animals, changes in histone acetylation show little correlation with the expression of TH response genes. On the other hand, in the intestine and tail, where TH response genes are known to be up-regulated more dramatically by TH than in most other organs, we demonstrate that TH treatment induces gene activation and histone H4 acetylation. These data argue for a role of histone acetylation in transcriptional regulation by TRs during amphibian development in some tissues, whereas in others changes in histone acetylation levels may play no or only a minor role, supporting the existence of important alternative mechanisms in gene regulation by TR.

Switch from Myc/Max to Mad1/Max binding and decrease in histone acetylation at the telomerase reverse transcriptase promoter during differentiation of HL60 cells

Recent evidence suggests that the Myc and Mad1 proteins are implicated in the regulation of the gene encoding the human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase. We have analyzed the in vivo interaction between endogenous c-Myc and Mad1 proteins and the hTERT promoter in HL60 cells with the use of the chromatin immunoprecipitation assay. The E-boxes at the hTERT proximal promoter were occupied in vivo by c-Myc in exponentially proliferating HL60 cells but not in cells induced to differentiate by DMSO. In contrast, Mad1 protein was induced and bound to the hTERT promoter in differentiated HL60 cells. Concomitantly, the acetylation of the histones at the promoter was significantly reduced. These data suggest that the reciprocal E-box occupancy by c-Myc and Mad1 is responsible for activation and repression of the hTERT gene in proliferating and differentiated HL60 cells, respectively. Furthermore, the histone deacetylase inhibitor trichostatin A inhibited deacetylation of histones at the hTERT promoter and attenuated the repression of hTERT transcription during HL60 cell differentiation. In addition, trichostatin A treatment activated hTERT transcription in resting human lymphocytes and fibroblasts. Taken together, these results indicate that acetylation/deacetylation of histones is operative in the regulation of hTERT expression.

Moderate increase in histone acetylation activates the mouse mammary tumor virus promoter and remodels its nucleosome structure.

The mouse mammary tumor virus (MMTV) promoter is regulated by steroid hormones through a hormone-responsive region that is organized in a positioned nucleosome. Hormone induction leads to a structural change of this nucleosome which makes its DNA more sensitive to cleavage by DNase I and enables simultaneous binding of all relevant transcription factors. In cells carrying either episomal or chromosomally integrated MMTV promoters, moderate acetylation of core histones, generated by treatment with low concentrations of the histone deacetylase inhibitors sodium butyrate or trichostatin A, enhances transcription from the MMTV promoter in the absence of hormone and potentiates transactivation by either glucocorticoids or progestins. At higher concentrations, histone deacetylase inhibitors reduce basal and hormone induced MMTV transcription. Inducing inhibitor concentrations lead to the same type of nucleosomal DNase I hypersensitivity as hormone treatment, suggesting that moderate acetylation of core histone activates the MMTV promoter by mechanisms involving chromatin remodeling similar to that generated by the inducing hormones.

Antiproliferative and phenotype-transforming antitumor agents derived from cysteine

Selective destruction of malignant tumor cells without damaging normal cells is an important goal for cancer chemotherapy in the 21st century. Differentiating agents that transform cancer cells to either a nonproliferating or normal phenotype could potentially be tissue-specific and avoid side effects of current drugs. However, most compounds that are presently known to differentiate cancer cells are histone deacetylase inhibitors that are of low potency or suffer from low bioavailability, rapid metabolism, reversible differentiation, and nonselectivity for cancer cells over normal cells. Here we describe 36 nonpeptidic compounds derived from a simple cysteine scaffold, fused at the C-terminus to benzylamine, at the N-terminus to a small library of carboxylic acids, and at the S-terminus to 4-butanoyl hydroxamate. Six compounds were cytotoxic at nanomolar concentrations against a particularly aggressive human melanoma cell line (MM96L), four compounds showed selectivities of greater than or equal to5:1 for human melanoma over normal human cells (NFF), and four of the most potent compounds were further tested and found to be cytotoxic for six other human cancer cell lines (melanomas SK-MEL-28, DO4; prostate DU145; breast MCF-7; ovarian JAM, CI80-13S). The most active compounds typically caused hyperacetylation of histones, induced p21 expression, and reverted phenotype of surviving tumor cells to a normal morphology. Only one compound was given orally at 5 mg/kg to healthy rats to look for bioavailaiblity, and it showed reasonably high levels in plasma (C-max 6 mug/mL, T-max 15 min) for at least 4 h. Results are sufficiently promising to support further work on refining this and related classes of compounds to an orally active, more tumor-selective, antitumor drug.

Exploiting novel cell cycle targets in the development of anticancer agents

In this review we provide a brief background on the cell cycle and then focus on two novel and emerging areas of cell cycle research that may prove to have significant relevance to the development of novel anticancer agents. In particular, we review the emerging evidence to suggest that histone deacetylase inhibitors may possess cancer cell-specific cytotoxicity due to their ability to target a novel G2/M checkpoint. We also review the recent literature supporting the proposition that inhibition of E2F activity in epithelial cancer cells may prove to be a useful differentiation therapy that operates via cell cycle-dependent and cell cycle-independent mechanisms.

Design, Synthesis, Potency and Cytoselectivity of Anticancer Agents Derived by Parallel Synthesis from alpha-Aminosuberic Acid

Chemotherapy in the last century was characterized by cytotoxic drugs that did not discriminate between cancerous and normal cell types and were consequently accompanied by toxic side effects that were often dose limiting. The ability of differentiating agents to selectively kill cancer cells or transform them to a nonproliferating or normal phenotype could lead to cell- and tissue-specific drugs without the side effects of current cancer chemotherapeutics. This may be possible for a new generation of histone deacetylase inhibitors derived from amino acids. Structure-activity relationships are now reported for 43 compounds derived from 2-aminosuberic acid that kill a range of cancer cells, 26 being potent cytotoxins against MM96L melanoma cells (IC50 20 nM-1 mu M), while 17 were between 5- and 60-fold more selective in killing MM96L melanoma cells versus normal (neonatal foreskin fibroblasts, NFF) cells. This represents a 10- to 100-fold increase in potency and up to a 10-fold higher selectivity over previously reported compounds derived from cysteine (J. Med. Chem. 2004, 47, 2984). Selectivity is also an underestimate, because the normal cells, NFF, are rarely all killed by the drugs that also induce selective blockade of the cell cycle for normal but not cancer cells. Selected compounds were tested against a panel of human cancer cell lines (melanomas, prostate, breast, ovarian, cervical, lung, and colon) and found to be both selective and potent cytotoxins (IC50 20 nM-1 mu M). Compounds in this class typically inhibit human histone deacetylases, as evidenced by hyperacetylation of histones in both normal and cancer cells, induce expression of p21, and differentiate surviving cancer cells to a nonproliferating phenotype. These compounds may be valuable leads for the development of new chemotherapeutic agents.

A small molecule activator of p300/CBP histone acetyltransferase promotes survival and neurite growth in a cellular model of Parkinson’s disease

Parkinson’s disease (PD) is a progressive neurodegenerative disease characterised by motor and non-motor symptoms, resulting from the degeneration of nigrostriatal dopaminergic neurons and peripheral autonomic neurons. Given the limited success of neurotrophic factors in clinical trials, there is a need to identify new small molecule drugs and drug targets to develop novel therapeutic strategies to protect all neurons that degenerate in PD. Epigenetic dysregulation has been implicated in neurodegenerative disorders, while targeting histone acetylation is a promising therapeutic avenue for PD. We and others have demonstrated that histone deacetylase inhibitors have neurotrophic effects in experimental models of PD. Activators of histone acetyltransferases (HAT) provide an alternative approach for the selective activation of gene expression, however little is known about the potential of HAT activators as drug therapies for PD. To explore this potential, the present study investigated the neurotrophic effects of CTPB (N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide), which is a potent small molecule activator of the histone acetyltransferase p300/CBP, in the SH-SY5Y neuronal cell line. We report that CTPB promoted the survival and neurite growth of the SH-SY5Y cells, and also protected these cells from cell death induced by the neurotoxin 6-hydroxydopamine. This study is the first to investigate the phenotypic effects of the HAT activator CTPB, and to demonstrate that p300/CBP HAT activation has neurotrophic effects in a cellular model of PD.

Design, synthesis and antiproliferative activity of hydroxyacetamide derivatives against HeLa cervical carcinoma cell and breast cancer cell line

Purpose: To design and develop a new series of histone deacetylase inhibitors (FP1 - FP12) and evaluate their inhibitory activity against hydroxyacetamide (HDAC) enzyme mixture-derived HeLa cervical carcinoma cell and MCF-7. Methods: The designed molecules (FP1 - FP12) were docked using AUTODOCK 1.4.6. FP3 and FP8 showed higher interaction comparable to the prototypical HDACI. The designed series of 2-[[(3- Phenyl/substituted Phenyl-[4-{(4-(substituted phenyl)ethylidine-2-Phenyl-1,3-Imidazol-5-One}](-4H- 1,2,4-triazol-5-yl)sulfanyl]-N-hydroxyacetamide derivatives (FP1-FP12) was synthesized by merging 2- [(4-amino-3-phenyl-4H- 1, 2, 4-triazol-5-yl) sulfanyl]-N-hydroxyacetamide and 2-{[4-amino-3-(2- hydroxyphenyl)-4H-1,2, 4-triazol-5-yl]sulfanyl}-N hydroxyacetamide derivatives with aromatic substituted oxazolone. The biological activity of the synthesized molecule (FP1-FP12) was evaluated against HDAC enzyme mixture-derived HeLa cervical carcinoma cell and breast cancer cell line (MCF-7). Results: HDAC inhibitory activity of FP10 showed higher IC50 (half-maximal concentration inhibitory activity) of 0.09 μM, whereas standard SAHA molecule showed IC50 of 0.057 μM. On the other hand, FP9 exhibited higher GI50 (50 % of maximal concentration that inhibited cell proliferation) of 22.8 μM against MCF-7 cell line, compared with the standard, adriamycin, with GI50 of (-) 50.2 μM. Conclusion: Synthesis, spectral characterization, and evaluation of HDAC inhibition activity and in vitro anticancer evaluation of novel hydroxyacetamide derivatives against MCF-7 cell line have been achieved. The findings indicate the emergence of potentialanticancer compounds.

A novel corepressor, BCoR-L1, represses transcription through an interaction with CtBP

Corepressors play a crucial role in negative gene regulation and are defective in several diseases. BCoR is a corepressor for the BCL6 repressor protein. Here we describe and functionally characterize BCoR-L1, a homolog of BCoR. When tethered to a heterologous promoter, BCoR-L1 is capable of strong repression. Like other corepressors, BCoR-L1 associates with histone deacetylase (HDAC) activity. Specifically, BCoR-L1 coprecipitates with the Class II HDACs, HDAC4, HDAC5, and HDAC7, suggesting that they are involved in its role as a transcriptional repressor. BCoR-L1 also interacts with the CtBP corepressor through a CtBP-interacting motif in its amino terminus. Abrogation of the CtBP binding site within BCoR-L1 partially relieves BCoR-L1-mediated transcriptional repression. Furthermore, BCoR-L1 is located on the E-cadherin promoter, a known CtBP-regulated promoter, and represses the E-cadherin promoter activity in a reporter assay. The inhibition of BCoR-L1 expression by RNA-mediated interference results in derepression of E-cadherin in cells that do not normally express E-cadherin, indicating that BCoR-L1 contributes to the repression of an authentic endogenous CtBP target.

Prostacyclin synthase expression and epigenetic regulation in nonsmall cell lung cancer

BACKGROUND: Prostacyclin synthase (PGIS) metabolizes prostaglandin H(2), into prostacyclin. This study aimed to determine the expression profile of PGIS in nonsmall cell lung cancer (NSCLC) and examine potential mechanisms involved in PGIS regulation. METHODS: PGIS expression was examined in human NSCLC and matched controls by reverse transcriptase polymerase chain reaction (RT-PCR), Western analysis, and immunohistochemistry. A 204-patient NSCLC tissue microarray was stained for PGIS and cyclooxygenase 2 (COX2) expression. Staining intensity was correlated with clinical parameters. Epigenetic mechanisms underpinning PGIS promoter expression were examined using RT-PCR, methylation-specific PCR, and chromatin immunoprecipitation analysis. RESULTS: PGIS expression was reduced/absent in human NSCLC protein samples (P <.0001), but not mRNA relative to matched controls. PGIS tissue expression was higher in squamous cell carcinoma (P =.004) and in male patients (P <.05). No significant correlation of PGIS or COX2 expression with overall patient survival was observed, although COX2 was prognostic for short-term (2-year) survival (P <.001). PGIS mRNA expression was regulated by DNA CpG methylation and histone acetylation in NSCLC cell lines, with chromatin remodeling taking place directly at the PGIS gene. PGIS mRNA expression was increased by both demethylation agents and histone deacetylase inhibitors. Protein levels were unaffected by demethylation agents, whereas PGIS protein stability was negatively affected by histone deacetylase inhibitors. CONCLUSIONS: PGIS protein expression is reduced in NSCLC, and does not correlate with overall patient survival. PGIS expression is regulated through epigenetic mechanisms. Differences in expression patterns between mRNA and protein levels suggest that PGIS expression and protein stability are regulated post-translationally. PGIS protein stability may have an important therapeutic role in NSCLC. © 2011 American Cancer Society.

Advances in the systemic therapy of malignant pleural mesothelioma

Malignant pleural mesothelioma is an aggressive thoracic malignancy associated with exposure to asbestos, and its incidence is anticipated to increase during the first half of this century. Chemotherapy is the mainstay of treatment, yet sufficiently robust evidence to substantiate the current standard of care has emerged only in the past 5 years. This Review summarizes the evidence supporting the clinical activity of chemotherapy, discusses the use of end points for its assessment and examines the influence of clinical and biochemical prognostic factors on the natural history of malignant pleural mesothelioma. Early-phase clinical trials of second-line and novel agents are emerging from an increased understanding of mesothelioma cell biology. Coupled with high-quality translational research, such developments have real potential to improve the outlook of patients at a time of increasing incidence.

Oxidative stress induced lung cancer and COPD : opportunities for epigenetic therapy

Reactive oxygen species (ROS) form as a natural by-product of the normal metabolism of oxygen and play important roles within the cell. Under normal circumstances the cell is able to maintain an adequate homeostasis between the formation of ROS and its removal through particular enzymatic pathways or via antioxidants. If however, this balance is disturbed a situation called oxidative stress occurs. Critically, oxidative stress plays important roles in the pathogenesis of many diseases, including cancer. Epigenetics is a process where gene expression is regulated by heritable mechanisms that do not cause any direct changes to the DNA sequence itself, and disruption of epigenetic mechanisms has important implications in disease. Evidence is emerging that histone deacetylases (HDACs) play decisive roles in regulating important cellular oxidative stress pathways including those involved with sensing oxidative stress and those involved with regulating the cellular response to oxidative stress. In particular aberrant regulation of these pathways by HDACs may play critical roles in cancer progression. In this review we discuss the current evidence linking epigenetics and oxidative stress and cancer, using chronic obstructive pulmonary disease and non-small cell lung cancer to illustrate the importance of epigenetics on these pathways within these disease settings. © 2009 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.