Targeting histone deacetylases for the treatment of disease.

The 'histone code' is a well-established hypothesis describing the idea that specific patterns of post-translational modifications to histones act like a molecular 'code' recognized and used by non-histone proteins to regulate specific chromatin functions. One modification, which has received significant attention, is that of histone acetylation. The enzymes that regulate this modification are described as lysine acetyltransferases or KATs, and histone deacetylases or HDACs. Due to their conserved catalytic domain HDACs have been actively targeted as a therapeutic target. The pro-inflammatory environment is increasingly being recognized as a critical element for both degenerative diseases and cancer. The present review will discuss the current knowledge surrounding the clinical potential and current development of histone deacetylases for the treatment of diseases for which a pro-inflammatory environment plays important roles, and the molecular mechanisms by which such inhibitors may play important functions in modulating the pro-inflammatory environment.

Histone acetylation/deacetylation and cancer: an "open" and "shut" case?

DNA in eukaryotic cells is packaged into chromatin. The main packaging component of chromatin is the nucleosome, and this is composed of proteins known as histones. Histones can be reversibly modified in several ways, and the best characterized of these modifications is histone acetylation. This is a reversible modification, which is carried out by two families of enzymes, the histone acetyltransferases (HATs), and the histone deacetylases (HDACs). These enzymes have important activities in many cellular processes including transcription, DNA replication and cell cycle progression. The mechanisms underlying tumor formation are multifaceted, and often involve mutations or alterations of genes involved with the regulation and control of the cell cycle or cell death. Because of their important roles in the regulation of such events, enzymes that affect histone acetylation status are increasingly being associated with tumors. This article describes some of the current knowledge about histone acetyltransferases and histone deacetylases, and how their multitudinal roles in cellular events may have important roles in tumorigensis.

Developmental-dependent DNA methylation of the IGF2 and H19 promoters is correlated to the promoter activities in human liver development.

We have previously shown that the four promoters of the IGF2 gene are under a tight but dynamic control during human liver development, whereby P3 and P1 are reciprocally active before and after birth respectively while the P2 and P4 promoters are constitutively active at a relatively lower level. In this study, we investigated the methylation status of the promoters P1 and P3 of IGF2 and the promoter region of the H19 gene in developing human livers ranging from fetal to late adult. A region of about 300 bp immediately upstream of the IGF2 exon 5 was found to be subjected to a developmental-specific methylation and this may correlate to the P3 promoter activity. The P1 domain of IGF2 was also found to be methylated in a developmentally-specific pattern. The promoter region of the H19 gene displayed different methylation patterns in different development stages showing decreased general methylation with increase of age. Therefore, regional- and developmental-specific DNA methylation is displayed in the promoter regions of the IGF2 and H19 genes. This may be an important factor involved in gene regulation in the developing human liver.

Expression levels of insulin-like growth factor binding proteins and insulin receptor isoforms in hepatoblastomas.

Hepatoblastoma, a rare pediatric liver tumour, is a poorly understood disease. While expression studies for some members of the Insulin-like growth factor axis have been studied in hepatoblastoma, a systematic analysis of the IGF-axis has not been carried out. We have examined a series of hepatoblastomas with matched normal liver tissue for gene expression differences with emphasis on members of the insulin-like growth factor binding proteins. The expression profiles obtained reveal that the expression of these genes are altered in these tumors. The results indicate that the IGF-axis is seriously disturbed in the tumors.

Microarray profiling of the effects of histone deacetylase inhibitors on gene expression in cancer cell lines.

Chromatin is a highly dynamic environment playing critical roles in the regulation of gene expression. Modifications to the proteins which make up the nucleosome core have been shown to have profound regulatory effects on gene expression. Of these, the best known modification is acetylation of the histone tails. Two enzymes regulate these processes, histone deacetylases and histone acetyltransferases. Both have been shown to have dysregulated functions in certain tumors. Several classes of histone deacetylase inhibitors have been isolated and are currently undergoing evaluation as potential therapeutic modalities in the treatment of cancer. In this study we examined the effects of three such inhibitors on general gene expression in three tumor cell lines derived from three separate tumor types using microarray gene profiling. Our results show that the patterns of alterations which emerge are similar for each cell type.

Differential expression of class I HDACs: roles of cell density and cell cycle.

Enzymes which affect histone acetylation status have been shown to play an important role in determining transcriptional activity in chromatin through conformational modification of its structure. Since the timely presence of such enzymes may be of critical importance, our experiments were designed to determine whether the level of expression of HDAC1 is cell cycle dependent and/or affected by a high cell density. Our results show that in mouse fibroblasts the expression of mHDAC1 is neither affected by cell cycle phases nor by cell density. In contrast, the expression of several hHDACs including hHDAC1 were affected in a cell density dependent fashion in the human prostate adenocarcinoma cell line PC3, paralleling our previously published findings in the hepatocellular carcinoma derived cell line Hep3B. Differential recruitment of HDAC mRNAs suggests that these enzymes may play unique roles in different cell types and under different environmental conditions (i.e., exposure to various cell densities and cell-cell contacts). Our study has implications for the proposed use of HDAC inhibitors in the treatment of human malignancy, highlighting issues of drug action selectivity in tissues and potential secondary effects.

Promoter-specific transcription of the IGF2 gene: a novel rapid, non-radioactive and highly sensitive protocol for mRNA analysis.

The human insulin-like growth factor-II (IGF2) is a regulatory peptide which is critical in normal fetal growth. IGF2 gene transcription is controlled by the usage of four promoters P1-P4 of which promoters P2-P4 are genomically imprinted. Disruption of imprinting and the resulting increase of gene dosage have been shown to be implicated in tumor progression in a variety of human tumors. Due to the need for high amounts of tissue material for conventional methods such as Northern blotting or ribonuclease protection assay (RPA), studies on IGF2 expression have most often been limited to the detection of total IGF2 transcript, though different dysregulatory events can be responsible for the abundance of IGF2 mRNA found in many tumors. We established a highly sensitive competitive RT-PCR assay for the four different transcripts of the IGF2 gene with transcript-specific external RNA competitors in which we take advantage of fluorescence-based quantification on a semiautomated sequencer. The amount of total RNA needed is approximately 100 times lower than the amounts required for Northern blotting or RPA, so that even cytological samples can be analyzed. We applied the assay to a series of eleven hepatoblastomas (HB) in which normal adjacent liver tissue could also be analyzed.

Modulation of splicing events in histone deacetylase 3 by various extracellular and signal transduction pathways.

Within the context of the chromatin environment histone deacetylases are important transcriptional regulators. Three classes of human histone deacetylases have currently been identified on the basis of their similarity to yeast proteins. The class I enzymes contain four members: HDACs 1-3 and HDAC8. Of these, HDAC3 is known to generate transcript variants with altered amino-terminal regions. Here we describe the identification of a novel splice variant of HDAC3, in which exon 3 is alternatively spliced from the messenger RNA transcript. We show that this human HDAC3 splice transcript is upregulated by treatments with histone deacetylase inhibitors. We also demonstrate evidence of splicing events in murine HDAC3 as a response to various signals, including switching between splice transcript isoforms following treatments with kinase inhibitors or by osmotic shock. In contrast, such switching events were not observed in human cells. These results indicate that differential pathways in mouse and human may control the regulation of HDAC3, and that splice variants may play important roles in responding to exogenous stimuli that act via signal transduction pathways.

Methylation, gene expression and the chromatin connection in cancer (review)

The mechanisms by which cells regulate gene expression are often altered in tumors. Modulating aspects of the components responsible for the nuclear packaging of DNA is one means by which the cell can control transcription, either by packaging the DNA such that access to specific sites of transcription is blocked or by modifying the DNA itself to prevent transcription factor binding. One such DNA modification is the methylation of cytosines. In addition, histone acetylation status has been linked recently through a large number of studies to the regulation of gene expression. Expressed genes are located in highly acetylated chromatin. The acetylation status of nucleosomes (the basic packaging unit of chromatin), is regulated by a group of enzymes, histone acetyltransferases (HATs), and histone deacetylases (HDACs). These two elements, methylation and histone acetylation have also been linked together, whereby methylation is used to direct gene repression through a histone deacetylase complex. Methylation, HATs, and HDACs have been shown to be altered in tumors. We present an overview of the current knowledge surrounding these elements in cancer, and in the final sections describe the likelihood of alterations of the histone modifying apparatus in hepatocellular carcinoma.

Modulating IGFBP-3 expression by trichostatin A: potential therapeutic role in the treatment of hepatocellular carcinoma.

The Hep3B cell line analyzed in the present study is a widely used in vitro model in studies characterizing pathogenetic, functional, and therapeutic aspects of human hepatocellular carcinoma (HCC). Here we have determined the chromosomal composition using a combination of cytogenetic techniques. In agreement with the original description for this cell line, Hep3B was found to have a hypotriploid chromosome content carrying 59-63 chromosomes and no cytogenetic differences were demonstrated between early and late passages suggesting that this cell line has remained stable after repeated subculturing. Mutations and alterations of the IGF-axis as well as of chromosome 1p34, where the genes for histone deacetylase 1 (HDAC1) and transforming growth factor beta receptor interacting protein-1 (TRIP-1) map, are frequent events in hepatocarcinogenesis. This study characterizes the Hep3B cell line in detail at the karyotypic level, using comparative genomic hybridization (CGH), spectral karyotyping (SKY), G-banding and FISH techniques. We have also examined the effects of the histone deacetylase inhibitor trichostatin A (TSA) on members of the IGF-axis, and analysed them with regard to the karyotype. The results show that expression of one member of the IGF-axis, IGFBP-3, is greatly upregulated by treatment of Hep3B cells with TSA. As IGFBP-3 has been shown to induce apoptosis, these results suggest a possible use for histone deacetylase inhibitors and/or IGFBP-3 in the treatment of HCC.

Expression of genes involved with cell cycle control, cell growth and chromatin modification are altered in hepatoblastomas.

Hepatoblastoma is a rare pediatric liver tumor. While much progress has been made in the treatment of the disease, very little is known about the moleculer events underlying the pathogenesis of this disease. We sought to investigate a series of hepatoblastomas for alterations in gene expression patterns with emphasis on important cell regulatory genes, including chromatin modifying enzymes, cyclin dependent kinase inhibitors, growth factors, oncogenes and cell cycle regulators. Total RNA was extracted from a series of sporadic hepatoblastomas with matched normal liver, some unmatched tumors and fetal livers, and gene expression was measured for various genes using RNase Protection Analysis (RPA). The results of this analysis show that the expression of many important regulatory genes are distinctly altered in these tumors, and a subset of tumors can be distinguished on the basis of these gene expression differences and histopathological features. Because the molecular events underlying the pathogenesis of this rare tumor are so poorly understood, this study represents a first step in determining some of the possible mechanisms involved which may provide future avenues of research.

Estrogen receptor ß activation impairs mitochondrial oxidative metabolism and affects malignant mesothelioma cell growth in vitro and in vivo.

Estrogen receptor (ER)-ß has been shown to possess a tumor suppressive effect, and is a potential target for cancer therapy. Using gene-expression meta-analysis of human malignant pleural mesothelioma, we identified an ESR2 (ERß coding gene) signature. High ESR2 expression was strongly associated with low succinate dehydrogenase B (SDHB) (which encodes a mitochondrial respiratory chain complex II subunit) expression. We demonstrate that SDHB loss induced ESR2 expression, and that activated ERß, by over-expression or by selective agonist stimulation, negatively affected oxidative phosphorylation compromising mitochondrial complex II and IV activity. This resulted in reduced mitochondrial ATP production, increased glycolysis dependence and impaired cell proliferation. The observed in vitro effects were phenocopied in vivo using a selective ERß agonist in a mesothelioma mouse model. On the whole, our data highlight an unforeseen interaction between ERß-mediated tumor suppression and energy metabolism that may be exploited to improve on the therapy for clinical management of malignant mesothelioma.

Targeting histone deacetylases for the treatment of immune, endocrine & metabolic disorders.

The 'histone code' is a well-established hypothesis describing the idea that specific patterns of post-translational modifications to histones act like a molecular "code" recognised and used by non-histone proteins to regulate specific chromatin functions. One modification which has received significant attention is that of histone acetylation. The enzymes which regulate this modification are described as histone acetyltransferases or HATs, and histone deacetylases or HDACs [1]. Due to their conserved catalytic domain HDACs have been actively targeted as a therapeutic target. The pro-inflammatory environment is increasingly being recognised as a critical element for both degenerative diseases and cancer. The present review will discuss the current knowledge surrounding the clinical potential & current development of histone deacetylases for the treatment of diseases for which a proinflammatory environment plays important roles, and the molecular mechanisms by which such inhibitors may play important functions in modulating the proinflammatory environment.

Histone deacetylase inhibitors target diabetes via chromatin remodeling or as chemical chaperones?

Globally, obesity and diabetes (particularly type 2 diabetes) represents a major challenge to world health. Despite decades of intense research efforts, the genetic basis involved in diabetes pathogenesis & conditions associated with obesity are still poorly understood. Recent advances have led to exciting new developments implicating epigenetics as an important mechanism underpinning diabetes and obesity related disease. One epigenetic mechanism known as the "histone code" describes the idea that specific patterns of post-translational modifications to histones act like a molecular "code" recognised and used by non-histone proteins to regulate specific chromatin functions. One modification which has received significant attention is that of histone acetylation. The enzymes which regulate this modification are described as lysine acetyltransferases or KATs and histone deacetylases or HDACs. Due to their conserved catalytic domain HDACs have been actively targeted as a therapeutic target. Some of the known inhibitors of HDACs (HDACi) have also been shown to act as "chemical chaperones" to alleviate diabetic symptoms. In this review, we discuss the available evidence concerning the roles of HDACs in regulating chaperone function and how this may have implications in the management of diabetes.

Effects of cell density and trichostatin A on the expression of HDAC1 and p57Kip2 in Hep 3B cells.

The interplay between the acetylation and deacetylation activities within the cell has been postulated to be a mechanism by which the cell regulates expression from genes at the level of chromatin. We have examined the expression pattern of the human histone deacetylase gene HDAC1 and the cyclin dependent kinase inhibitor p57Kip2 in the hepatocellular carcinoma cell line Hep 3B. HDAC1 expression was elevated at low cell densities, but once a critical threshold point in cell density was attained, expression was reduced to very low levels. Treatment of the cells with trichostatin A (TSA), a potent inhibitor of histone deacetylases, was found to affect expression. p57Kip2 was found to be downregulated by TSA, whereas HDAC1 was upregulated. These effects were found to be cell density dependent. The results suggest that HDAC1 plays a role in its own regulation, and that investigations using TSA should be carried out when cells grow exponentially.

The human histone deacetylase family.

Since the identification of the first histone deacetylase (Taunton et al., Science 272, 408-411), several new members have been isolated. They can loosely be separated into entities on the basis of their similarity to various yeast histone deacetylases. The first class is represented by its closeness to the yeast Rpd3-like proteins, and the second most recently discovered class has similarities to yeast Hda1-like proteins. However, due to the fact that several different research groups isolated the Hda1-like histone deacetylases independently, there have been various different nomenclatures used to describe the various members, which can lead to confusion in the interpretation of this family's functions and interactions. With the discovery of another novel murine histone deacetylase, homologous to yeast Sir2, the number of members of this family is set to increase, as 7 human homologues of this gene have been isolated. In the light of these recent discoveries, we have examined the literature data and conducted a database analysis of the isolated histone deacetylases and potential candidates. The results obtained suggest that the number of histone deacetylases within the human genome may be as high as 17 and are discussed in relation to their homology to the yeast histone deacetylases.

The insulin-like growth factors and insulin-signalling systems: an appealing target for breast cancer therapy?

There is compelling evidence from epidemiological studies in humans, as well as in vitro and in vivo experimental observations including transgenic animal models, for a role of the IGF/insulin signalling system in cancer tumourigenesis. In this review focused on breast cancer, we review the experimental evidence, discuss the cellular and molecular mechanisms of tumourigenicity by the IGFs and insulin and various possible therapeutic strategies based on the mechanisms discussed.