Histone deacetylases (HDAC) modulate acetylation and the function of histone and non-histone proteins. HDAC inhibitors have been developed to block the aberrant action of HDACs in cancer, and several are in clinical use (vorinostat, romidepsin, and valproic acid). Detailed understanding of their action is lacking, however, and their clinical activity is limited in most cases. Recently, HDACs have been involved in the control of the DNA damage response (DDR) at several levels and in directly regulating the acetylation of a number of DDR proteins (including CtIP and Exo1). Mechanistically, acetylation leads to the degradation of double-strand break repair enzymes through autophagy, providing a novel, direct link between DDR and autophagy. These observations, obtained in yeast cells, should now be translated to mammalian model systems and cancer cells to reveal whether this acetylation link is maintained in mammals, and if and how it is deregulated in cancer. In addition to HDACs, DDR and autophagy have been addressed pharmacologically, suggesting that the acetylation link, if involved in cancer, can be exploited for the design of new anticancer treatments. Clin Cancer Res; 18(9); 1-7. ©2012 AACR.

Histone deacetylase (HDAC) inhibitors are clinically relevant because they are used as anticancer drugs. Recent evidence sheds light on an intriguing connection among the DNA damage response (DDR), protein acetylation, and autophagy. HDAC inhibitors have been shown to counteract key steps in the cellular response to double-strand break formation by affecting checkpoint activation, homologous recombination-mediated repair of DNA lesions, and stability of crucial enzymes involved in resection of DNA ends. The degradation of the resection factors depends on autophagy, which plays a detrimental role when cells are in a hyperacetylated state and experience treatment with radiomimetic anticancer drugs. Future work will be required to further investigate the mechanisms underlying the link between acetylation, autophagy, and the DDR, as well as the significance of mTORC1 inhibitors, which are potent inducers of autophagy that are now used in cancer treatment. Cancer Res; 72(6); 1332-5. ©2012 AACR.

A series of spiro[chromane-2,4'-piperidine] derivatives based on a previously published lead benzyl spirocycle 1 and bearing various N-aryl and N-alkylaryl substituents on the piperidine ring were prepared as novel histone deacetylase (HDAC) inhibitors. The compounds were evaluated for their abilities to inhibit nuclear HDACs, their in vitro antiproliferative activities, and in vitro ADME profiles. Based on these activities, 4-fluorobenzyl and 2-phenylethyl spirocycles were selected for further characterization. In vivo pharmacokinetic (PK) studies showed that both compounds exhibit an overall lower clearance rate, an increased half-life, and higher AUCs after intravenous and oral administration than spiropiperidine 1 under the conditions used. The improved PK behavior of these two compounds also correlated with superior in vivo antitumor activity in an HCT-116 xenograft model.

Aberrant histone acetylation was physiopathologically associated with the development of acute myeloid leukemias (AMLs). Reversal of histone deacetylation by histone deacetylase inhibitor (HDACis) activates a cell death program that allows tumor regression in mouse models of AMLs. We have used several models of PML-RARA-driven acute promyelocytic leukemias (APLs) to analyze the in vivo effects of valproic acid, a well-characterized HDACis. Valproic acid (VPA)-induced rapid tumor regression and sharply prolonged survival. However, discontinuation of treatment was associated to an immediate relapse. In vivo, as well as ex vivo, VPA-induced terminal granulocytic differentiation. Yet, despite full differentiation, leukemia-initiating cell (LIC) activity was actually enhanced by VPA treatment. In contrast to all-trans retinoic acid (ATRA) or arsenic, VPA did not degrade PML-RARA. However, in combination with ATRA, VPA synergized for PML-RARA degradation and LIC eradication in vivo. Our studies indicate that VPA triggers differentiation, but spares LIC activity, further uncouple differentiation from APL clearance and stress the importance of PML-RARA degradation in APL cure.Leukemia advance online publication, 6 March 2012; doi:10.1038/leu.2012.39.

Formalin-fixed, paraffin-embedded (FFPE) samples represent the gold standard for storage of pathology samples. Here we describe pathology tissue chromatin immunoprecipitation (PAT-ChIP), a technique for extraction and high-throughput analysis, by techniques such as ChIP-seq, of chromatin derived from FFPE samples. Technically, the main challenge of PAT-ChIP is the preparation of good-quality chromatin from FFPE samples. Here we provide a detailed explanation of the methodology used, the choice of reagents and the troubleshooting steps required to establish a robust chromatin preparation procedure. Other steps have also been adapted from existing techniques to optimize their use for PAT-ChIP-seq. The protocol requires 4 d from the start to the end of the PAT-ChIP procedure. PAT-ChIP provides, for the first time, the chance to perform analyses of histone modifications and transcription factor binding on a genome-wide scale using patient-derived FFPE samples. This technique therefore allows the immediate use of pathology archives (even those that are several years old) for epigenetic analyses and the identification of candidate epigenetic biomarkers or targets.

ABSTRACT: BACKGROUND: High-throughput sequencing is generating massive amounts of data at a pace that largely exceeds the throughput of data analysis routines. Here we introduce Fish the ChIPs (FC), a computational pipeline aimed at a broad public of users and designed to perform complete ChIP-Seq data analysis of an unlimited number of samples, thus increasing throughput, reproducibility and saving time. RESULTS: Starting from short read sequences, FC performs the following steps: 1) quality controls, 2) alignment to a reference genome, 3) peak calling, 4) genomic annotation, 5) generation of raw signal tracks for visualization on the UCSC and IGV genome browsers. FC exploits some of the fastest and most effective tools today available. Installation on a Mac platform requires very basic computational skills while configuration and usage are supported by a user-friendly graphic user interface. Alternatively, FC can be compiled from the source code on any Unix machine and then run with the possibility of customizing each single parameter through a simple configuration text file that can be generated using a dedicated user-friendly web-form. Considering the execution time, FC can be run on a desktop machine, even though the use of a computer cluster is recommended for analyses of large batches of data. FC is perfectly suited to work with data coming from Illumina Solexa Genome Analyzers or ABI SOLiD and its usage can potentially be extended to any sequencing platform. CONCLUSIONS: Compared to existing tools, FC has two main advantages that make it suitable for a broad range of users. First of all, it can be installed and run by wet biologists on a Mac machine. Besides it can handle an unlimited number of samples, being convenient for large analyses. In this context, computational biologists can increase reproducibility of their ChIP-Seq data analyses while saving time for downstream analyses. Reviewers This article was reviewed by Gavin Huttley, George Shpakovski and Sarah Teichmann.

Melatonin, nocturnally secreted by the pineal gland, regulates a variety of physiological functions, including reproduction. Here, we investigated the evidence of melatonin binding sites in frog tissue (brain, retina, and testis) through saturation and competition binding experiments. In the frog, Pelophylax esculentus, our results confirm the presence of a single class of melatonin-specific binding sites in the brain and retina, but not in the testis. Further experiments have been done using biomolecular approaches (PCR analysis). Here, we report the isolation of a cDNA encoding for a melatonin receptor type (mel1b) from brain, retina, and testis of the P. esculentus. PCR analysis revealed that melatonin expression is higher in the brain and retina, whereas it is lower in the testis. The presence of a melatonin receptor transcript in the frog testis corroborates our previous results obtained in in vitro experiments that suggest that melatonin might act directly in male vertebrate gonads, and indicates that the frog testis may be a suitable model to verify the role of indolamine in testicular activity. J. Exp. Zool. 313A, 2011. © 2011 Wiley-Liss, Inc.

Mast cells are best known as multifunctional entities that may confer a benefit on immune system. This review presents the known facts on mast-cell system and breakthroughs in mast-cell biology in fish, amphibians, reptiles, and birds. As compared to mammals, there are relatively few data available on mast cells in many nonmammalian vertebrates. Nevertheless, like in mammals, mast cells in nonmammalian vertebrates contain a wide range of bioactive compounds including histamine, heparin, neuropeptides, and neutral proteases. In bony fishes, these cells secrete antimicrobial peptides as well. Mast cells have a widespread distribution in the brain, endocrine glands, intestine, liver, kidney, skin, tongue, and lungs, the highest concentration occurring in different tissues in the different taxa. Currently, researchers are grappling with the nature of scientific support to substantiate the functional importance of mast cells in nonmammalian vertebrates. Ultimately, the origin and evolution of vertebrate mast cell is of great interest to comparative immunologists seeking an underlying trend in the phylogenetic development of immunity.

New spiro[chromane-2,4'-piperidine] and spiro[benzofuran-2,4'-piperidine] hydroxamic acid derivatives as HDAC inhibitors have been identified by combining privileged structures with a hydroxamic acid moiety as zinc binding group. The compounds were evaluated for their ability to inhibit nuclear extract HDACs and for their in vitro antiproliferative activity on different tumor cell lines. This work resulted in the discovery of spirocycle 30d that shows good oral bioavailability and tumor growth inhibition in an HCT-116 murine xenograft model.