Mirco Fanelli

Chromatin immunoprecipitation and high-throughput sequencing from paraffin-embedded pathology tissue

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.

Pathology tissue–chromatin immunoprecipitation, coupled with high-throughput sequencing, allows the epigenetic profiling of patient samples

Epigenetic alterations in the pattern of DNA and histone modifications play a crucial role in cancer development. Analysis of patient samples, however, is hampered by technical limitations in the study of chromatin structure from pathology archives that usually consist of heavily fixed, paraffin-embedded material. Here, we present a methodology [pathology tissue–ChIP (PAT-ChIP)] to extract and immunoprecipitate chromatin from paraffin-embedded patient samples up to several years old. In a pairwise comparison with canonical ChIP, PAT-ChIP showed a high reproducibility of results for several histone marks and an identical ability to detect dynamic changes in chromatin structure upon pharmacological treatment. Finally, we showed that PAT-ChIP can be coupled with high-throughput sequencing (PAT-ChIP-Seq) for the genome-wide analysis of distinct chromatin modifications. PAT-ChIP therefore represents a versatile procedure and diagnostic tool for the analysis of epigenetic alterations in cancer and potentially other diseases.

Synaptogenesis in adult-generated hippocampal granule cells is affected by behavioral experiences

Adult‐generated hippocampal immature neurons play a functional role after integration in functional circuits. Previously, we found that hippocampus‐dependent learning in Morris water maze affects survival of immature neurons, even before they are synaptically contacted. Beside learning, this task heavily engages animals in physical activity in form of swimming; physical activity enhances hippocampal neurogenesis. In this article, the effects of training in Morris water maze apparatus on the synapse formation onto new neurons in hippocampus dentate gyrus and on neuronal maturation were investigated in adult rats. Newborn cells were identified using retroviral GFP‐expressing virus infusion. In the first week after virus infusion, rats were trained in Morris water maze apparatus in three different conditions (spatial learning, cue test, and swimming). Properties of immature neurons and their synaptic response to perforant pathway stimulation were electrophysiologically investigated early during neuronal maturation. In controls, newborn cells showing GABAergic and glutamatergic responses were found for the first time at 8 and 10 days after mitosis, respectively; no cell with glutamatergic response only was found. Twelve days after virus infusion almost all GFP‐positive cells showed both synaptic responses. The main result we found was the anticipated appearance of GABAergic synapses at 6 days in learner, cued and swimmer rats, supported also by immunohistochemical result. Swimmer rats showed the highest percentage of GFP‐positive neurons with glutamatergic response at 10 and 12 days postmitosis. Moreover, primary dendrites were more numerous at 7 days in learner, cued and swimmer rats and swimmer rats showed the greatest dendritic tree complexity at 10 days. Finally, voltage‐dependent Ca2+ current was found in a larger number of newborn neurons at 7 days postinfusion in learner, cued and swimmer rats. In conclusion, experiences involving physical activity contextualized in an exploring behavior affect synaptogenesis in adult‐generated cells and their early stages of maturation.

DNA Demethylating Antineoplastic Strategies: A Comparative Point of View

Despite the involvement of genetic alterations in neoplastic cell transformation, it is increasingly evident that abnormal epigenetic patterns, such as those affecting DNA methylation and histone posttranslational modifications (PTMs), play an essential role in the early stages of tumor development. This finding, together with the evidence that epigenetic changes are reversible, enabled the development of new antineoplastic therapeutic approaches known as epigenetic therapies. Epigenetic modifications are involved in the control of gene expression, and their aberrant distribution is thought to participate in neoplastic transformation by causing the deregulation of crucial cellular pathways. Epigenetic drugs are able to revert the defective gene expression profile of cancer cells and, consequently, reestablish normal molecular pathways. Considering the emerging interest in epigenetic therapeutics, this review focuses on the approaches affecting DNA methylation, evaluates novel strategies and those already approved for clinical use, and compares their therapeutic potential.

Malten, a new synthetic molecule showing in vitro antiproliferative activity against tumour cells and induction of complex DNA structural alterations

Background:Hydroxypyrones represent several classes of molecules known for their high synthetic versatility. This family of molecules shows several interesting pharmaceutical activities and is considered as a promising source of new antineoplastic compounds.Methods:In the quest to identify new potential anticancer agents, a new maltol (3-hydroxy-2-methyl-4-pyrone)-derived molecule, named malten (N,N′-bis((3-hydroxy-4-pyron-2-yl)methyl)-N,N′-dimethylethylendiamine), has been synthesised and analysed at both biological and molecular levels for its antiproliferative activity in eight tumour cell lines.Results:Malten exposure led to a dose-dependent reduction in cell survival in all the neoplastic models studied. Sublethal concentrations of malten induce profound cell cycle changes, particularly affecting the S and/or G2-M phases, whereas exposure to lethal doses causes the induction of programmed cell death. The molecular response to malten was also investigated in JURKAT and U937 cells. It showed the modulation of genes having key roles in cell cycle progression and apoptosis. Finally, as part of the effort to clarify the action mechanism, we showed that malten is able to impair DNA electrophoretic mobility and drastically reduce both PCR amplificability and fragmentation susceptibility of DNA.Conclusion:Taken together, these results show that malten may exert its antiproliferative activity through the induction of complex DNA structural modifications. This evidence, together with the high synthetic versatility of maltol-derived compounds, makes malten an interesting molecular scaffold for the future design of new potential anticancer agents.

Physical exercise and environment exploration affect synaptogenesis in adult-generated neurons in the rat dentate gyrus: Possible role of BDNF

A brief training in a pool maze, with or without cognitive tasks, modifies the synaptogenesis and maturation of newborn neurons in adult rat dentate gyrus. These types of trainings have many aspects, including physical activity and exploration. Therefore, to evaluate whether physical exercise and environment exploration are able to affect synapse formation and the maturation of adult-generated neurons, GFP-retrovirus infusion was performed on rats which, on the fourth day after injection, were housed under running conditions or allowed to explore an enriched environment briefly in the absence of exercise for the following three days. Afterward, at the end of the trainings, electrophysiological and morphological studies were conducted. Considering that neurotrophic factors increase after exercise or environment exploration, hippocampal BDNF levels and TrkB receptor activation were evaluated. In this study, we show that both spontaneous physical activity and enriched environment exploration induced synaptogenesis and T-type voltage-dependent Ca(2+) currents in very immature neurons. Hippocampal BDNF levels and TrkB receptor activation were determined to be increasing following physical activity and exploration. A possible contribution of BDNF signaling in mediating the observed effects was supported by the use of 7-8-dihydroxyflavone, a selective TrkB agonist, and of ANA-12, an inhibitor of TrkB receptors.

Polyphenol-rich strawberry extract (PRSE) shows in vitro and in vivo biological activity against invasive breast cancer cells

We describe the biological effects of a polyphenol-rich strawberry extract (PRSE), obtained from the “Alba” variety, on the highly aggressive and invasive basal-like breast cancer cell line A17. Dose-response and time-course experiments showed that PRSE is able to decrease the cellular viability of A17 cells in a time- and dose-dependent manner. PRSE effect on cell survival was investigated in other tumor and normal cell lines of both mouse and human origin, demonstrating that PRSE is more active against breast cancer cells. Cytofluorimetric analysis of A17 cells demonstrated that sub-lethal doses of PRSE reduce the number of cells in S phase, inducing the accumulation of cells in G1 phase of cell cycle. In addition, the migration of A17 cells was studied monitoring the ability of PRSE to inhibit cellular mobility. Gene expression analysis revealed the modulation of 12 genes playing different roles in the cellular migration, adhesion and invasion processes. Finally, in vivo experiments showed the growth inhibition of A17 cells orthotopically transplanted into FVB syngeneic mice fed with PRSE. Overall, we demonstrated that PRSE exerts important biological activities against a highly invasive breast cancer cell line both in vitro and in vivo suggesting the strawberry extracts as preventive/curative food strategy.

Multi-use NBD-based tetra-amino macrocycle: fluorescent probe for metals and anions and live cell marker.

Ligand L (4-(7-nitrobenzo[1,2,5]oxadiazole-4-yl)-1,7-dimethyl-1,4,7,10-tetra-azacyclododecane) is a versatile fluorescent sensor useful for Cu(II), Zn(II) and Cd(II) metal detection, as a building block of fluorescent metallo-receptor for halide detection, and as an organelle marker inside live cells. Ligand L undergoes a chelation-enhanced fluorescence (CHEF) effect upon metal coordination in acetonitrile solution. In all three complexes investigated the metal cation is coordinatively unsaturated; thus, it can bind secondary ligands as anionic species. The crystal structure of [ZnLCl](ClO(4)) is discussed. Cu(II) and Zn(II) complexes are quenched upon halide interaction, whereas the [CdL](2+) species behaves as an OFF-ON sensor for halide anions in acetonitrile solution. The mechanism of the fluorescence response in the presence of the anion depends on the nature of the metal ion employed and has been studied by spectroscopic methods, such as NMR spectroscopy, UV/Vis and fluorescence techniques and by computational methods. Subcellular localization experiments performed on HeLa cells show that L mainly localizes in spot-like structures in a polarized portion of the cytosol that is occupied by the Golgi apparatus to give a green fluorescence signal.

Modulating the Sensor Response to Halide Using NBD-Based Azamacrocycles

Ligand L (2,6-bis{[7-(7-nitrobenzo[1,2,5]oxadiazole-4-yl)-3,10-dimethyl-1,4,7,10-tetraazacyclododeca-1-yl]methyl}phenol) is a fluorescent sensor that is useful for detecting Cu(II), Zn(II), and Cd(II). Some of the complexes formed are able to sense the presence of halides in solution. L passes through the cellular membrane, becoming fluorescent inside cells. The H(-1)L- species is able to form dinuclear complexes with [M(2)H(-1)L]3+ stoichiometry with Cu(II), Zn(II), and Cd(II) ions, experiencing a CHEF effect upon metal coordination in an acetonitrile/water 95:5 (v/v) solution. In all three of the complexes investigated, the metal cations are coordinatively unsaturated and can therefore bind secondary ligands as anionic species. The crystal structure of [Cd(2)(H(-1)L)Cl(2)](ClO(4))·4H(2)O is discussed. The Zn(II) complex behaves as an OFF-ON sensor for fluoride and chloride anions.

PAT-ChIP coupled with laser microdissection allows the study of chromatin in selected cell populations from paraffin-embedded patient samples

BackgroundThe recent introduction of pathology tissue-chromatin immunoprecipitation (PAT-ChIP), a technique allowing chromatin immunoprecipitation from formalin-fixed and paraffin-embedded (FFPE) tissues, has expanded the application potential of epigenetic studies in tissue samples. However, FFPE tissue section analysis is strongly limited by tissue heterogeneity, which hinders linking the observed epigenetic events to the corresponding cellular population. Thus, ideally, to take full advantage of PAT-ChIP approaches, procedures able to increase the purity and homogeneity of cell populations from FFPE tissues are required.ResultsIn this study, we tested the use of both core needle biopsies (CNBs) and laser microdissection (LMD), evaluating the compatibility of these methods with the PAT-ChIP procedure. Modifications of the original protocols were introduced in order to increase reproducibility and reduce experimental time. We first demonstrated that chromatin can be prepared and effectively immunoprecipitated starting from 0.6-mm-diameter CNBs. Subsequently, in order to assess the applicability of PAT-ChIP to LMD samples, we tested the effects of hematoxylin or eosin staining on chromatin extraction and immunoprecipitation, as well as the reproducibility of our technique when using particularly low quantities of starting material. Finally, we carried out the PAT-ChIP using chromatin extracted from either normal tissue or neoplastic lesions, the latter obtained by LMD from FFPE lung sections derived from mutant K-rasv12 transgenic mice or from human adeno- or squamous lung carcinoma samples. Well characterized histone post-translational modifications (HPTMs), such as H3K4me3, H3K27me3, H3K27Ac, and H3K9me3, were specifically immunoselected, as well as the CTCF transcription factor and RNA polymerase II (Pol II).ConclusionsEpigenetic profiling can be performed on enriched cell populations obtained from FFPE tissue sections. The improved PAT-ChIP protocol will be used for the discovery and/or validation of novel epigenetic biomarkers in FFPE human samples.