Alice Pasini

Priming adult stem cells by hypoxic pretreatments for applications in regenerative medicine

The efficiency of regenerative medicine can be ameliorated by improving the biological performances of stem cells before their transplantation. Several ex-vivo protocols of non-damaging cell hypoxia have been demonstrated to significantly increase survival, proliferation and post-engraftment differentiation potential of stem cells. The best results for priming cultured stem cells against a following, otherwise lethal, ischemic stress have been obtained with brief intermittent episodes of hypoxia, or anoxia, and reoxygenation in accordance with the extraordinary protection afforded by the conventional maneuver of ischemic preconditioning in severely ischemic organs. These protocols of hypoxic preconditioning can be rather easily reproduced in a laboratory; however, more suitable pharmacological interventions inducing stem cell responses similar to those activated in hypoxia are considered among the most promising solutions for future applications in cell therapy. Here we want to offer an up-to-date review of the molecular mechanisms translating hypoxia into beneficial events for regenerative medicine. To this aim the involvement of epigenetic modifications, microRNAs, and oxidative stress, mainly activated by hypoxia inducible factors, will be discussed. Stem cell adaptation to their natural hypoxic microenvironments (niche) in healthy and neoplastic tissues will be also considered.

Organosulfur derivatives of the HDAC inhibitor valproic acid sensitize human lung cancer cell lines to apoptosis and to cisplatin cytotoxicity.

Lung cancer is the leading cause of cancer mortality worldwide and despite efforts made to improve clinical results, continuing poor survival rates indicate that novel therapeutic approaches are needed. Valproic acid (VPA), a short‐chain branched fatty acid used mainly for the treatment of epilepsy and bipolar disorder, has been shown to inhibit class I histone deacetylases (HDAC‐I), a group of enzymes involved in chromatin remodeling and which are thought to play a role in tumor development. Although evidence of VPAs therapeutic efficacy has also been observed in patients with solid tumors, the very high concentration required to induce antitumor activity limits its clinical usefulness. We used a panel of NSCLC cell lines to evaluate the activity and mechanisms of action of organosulfur valproic acid derivatives, a promising new class of compounds designed to improve the safety and efficacy of the valproic acid molecule and created by coupling it with a hydrogen sulfide (H2S)‐releasing moiety. Our results highlighted the increased cytotoxic activity of the novel organosulfur derivatives, ACS33 and ACS2, with respect to VPA, starting from low concentrations. In particular, ACS2 exhibited important pro‐apoptotic activity triggered by the mitochondrial pathway and also showed anti‐invasion potential. Furthermore, our in vitro results identified a highly effective combination schedule of ACS2 and cisplatin capable of inducing a synergistic interaction even when the two drugs were used at low concentrations, which could prove a valid alternative to traditional chemotherapeutic regimens used for advanced lung cancer. Further studies are needed to confirm these preliminary findings. J. Cell. Physiol. 227: 3389–3396, 2012.

Chromatin remodeling by polyamines and polyamine analogs

Natural polyamines are involved in many molecular processes, including maintenance of DNA structure and RNA processing and translation. Our aim here is to present an overview of the literature concerning the significance of polyamines in the modulation of chromatin arrangement and the transcriptional regulation of gene expression. The pleiotropic picture emerging from the published data highlights that these polycations take part in apparently diverging effects, possibly depending on the heterogeneous experimental settings described, and on a methodological approach aimed at the evaluation of the global levels of the histone chemical modifications. Since the relevant changes observed appear to be rather local and gene specific, investigating histone modifications at the level of specific gene promoters of interest is thus to be recommended for future studies. Furthermore, decoding the multiple regulatory mechanisms by which polyamines exert their influence on chromatin-modifier enzymes will reasonably require focus on selected individual polyamine-regulated genes. The evaluation of the many known chromatin-remodeling enzymes for their individual susceptibility to polyamines or polyamine derivatives will also be helpful: determining how they discriminate between the different enzyme isoforms is expected to be a fruitful line of research for drug discovery, e.g., in cancer prevention and therapy. Indeed, polyamine derivatives acting as epigenetic modulators appear to be molecules with great potential as antitumor drugs. All these novel polyamine-based pharmacologically active molecules are thus promising tools, both as a stand-alone strategy and in combination with other anticancer compounds.

Targeting Chromatin-Mediated Transcriptional Control of Gene Expression in Non-Small Cell Lung Cancer Therapy: Preclinical Rationale and Clinical Results

Targeting chromatin-mediated transcriptional control of gene expression is nowadays considered a promising new strategy, transcending conventional anticancer therapy. As a result, molecules acting as DNA demethylating agents or histone deacetylase inhibitors (HDACi) have entered the clinical arena in the last decade. Given the evidence suggesting that epigenetic regulation is significantly involved in lung cancer development and progression, the potential of epigenetically active compounds to modulate gene expression and reprogram cancer cells to a less aggressive phenotype is, at present, a promising strategy. Accordingly, a large number of compounds that interact with the epigenetic machinery of gene expression regulation are now being developed and tested as potential antitumor agents, either alone or in combination with standard therapy. The preclinical rationale and clinical data concerning the pharmacological modulation of chromatin organization in non-small cell lung cancer (NSCLC) is described in this review. Although preclinical data suggest that a pharmacological treatment targeting the epigenetic machinery has relevant activity over the neoplastic phenotype of NSCLC cells, clinical results are disappointing, leading only to short periods of disease stabilization in NSCLC patients. This evidence calls for a significant rethinking of strategies for an effective epigenetic therapy of NSCLC. The synergistic effect of concurrent epigenetic therapies, use at low doses, the priming of current treatments with previous epigenetic drugs, and the selection of clinical trial populations based on epigenetic biomarkers/signatures appear to be the cornerstones of a mature therapeutic strategy aiming to establish new regimens for reprogramming malignant cells and improving the clinical history of affected patients.

Experimental measurements and mathematical modeling of biological noise arising from transcriptional and translational regulation of basic synthetic gene circuits

The small number of molecules, unevenly distributed within an isogenic cell population, makes gene expression a noisy process, and strategies have evolved to deal with this variability in protein concentration and to limit its impact on cellular behaviors. As translational efficiency has a major impact on biological noise, a possible strategy to control noise is to regulate gene expression processes at the post-transcriptional level. In this study, fluctuations in the concentration of a green fluorescent protein were compared, at the single cell level, upon transformation of an isogenic bacterial cell population with synthetic gene circuits implementing either a transcriptional or a post-transcriptional control of gene expression. Experimental measurements showed that protein variability is lower under post-transcriptional control, when the same average protein concentrations are compared. This effect is well reproduced by stochastic simulations, supporting the hypothesis that noise reduction is due to the control mechanism acting on the efficiency of translation. Similar strategies are likely to play a role in noise reduction in natural systems and to be useful for controlling noise in synthetic biology applications.

Interplay between EZH2 and G9a Regulates CXCL10 Gene Repression in Idiopathic Pulmonary Fibrosis

&NA; Selective repression of the antifibrotic gene CXCL10 contributes to tissue remodeling in idiopathic pulmonary fibrosis (IPF). We have previously reported that histone deacetylation and histone H3 lysine 9 (H3K9) methylation are involved in CXCL10 repression. In this study, we explored the role of H3K27 methylation and the interplay between the two histone lysine methyltransferases enhancer of zest homolog 2 (EZH2) and G9a in CXCL10 repression in IPF. By applying chromatin immunoprecipitation, Re‐ChIP, and proximity ligation assays, we demonstrated that, like G9a‐mediated H3K9 methylation, EZH2‐mediated histone H3 lysine 27 trimethylation (H3K27me3) was significantly enriched at the CXCL10 promoter in fibroblasts from IPF lungs (F‐IPF) compared with fibroblasts from nonfibrotic lungs, and we also found that EZH2 and G9a physically interacted with each other. EZH2 knockdown reduced not only EZH2 and H3K27me3 but also G9a and H3K9me3, and G9a knockdown reduced not only G9 and H3K9me3 but also EZH2 and H3K27me3. Depletion and inhibition of EZH2 and G9a also reversed histone deacetylation and restored CXCL10 expression in F‐IPF. Furthermore, treatment of fibroblasts from nonfibrotic lungs with the profibrotic cytokine transforming growth factor‐&bgr;1 increased EZH2, G9a, H3K27me3, H3K9me3, and histone deacetylation at the CXCL10 promoter, similar to that observed in F‐IPF, which was correlated with CXCL10 repression and was prevented by EZH2 and G9a knockdown. These findings suggest that a novel and functionally interdependent interplay between EZH2 and G9a regulates histone methylation‐mediated epigenetic repression of the antifibrotic CXCL10 gene in IPF. This interdependent interplay may prove to be a target for epigenetic intervention to restore the expression of CXCL10 and other antifibrotic genes in IPF.

Novel Polyamine–Naphthalene Diimide Conjugates Targeting Histone Deacetylases and DNA for Cancer Phenotype Reprogramming

A series of hybrid compounds was designed to target histone deacetylases and ds-/G-quadruplex DNAs by merging structural features deriving from Scriptaid and compound 1. Compound 6 binds different DNA arrangements, inhibits HDACs both in vitro and in cells, and is able to induce a reduction of cell proliferation. Moreover, compound 6 displays cell phenotype-reprogramming properties since it prevents the epithelial to mesenchymal transition in cancer cells, inducing a less aggressive and migratory phenotype, which is one of the goals of present innovative strategies in cancer therapies.

Molecular mechanisms of ischemic preconditioning and postconditioning as putative therapeutic targets to reduce tumor survival and malignancy.

In tumors intermittent hypoxia has been reported to be more representative than normoxia or continuous exposure to low oxygen concentrations. Intermittent hypoxia is thought to increase tumor resistance against both anti-cancer therapy and the sustained ischemia that randomly occurs because of the dynamic nature of tumor vasculature. Here, we hypothesize that the molecular mechanisms underlying intermittent hypoxia in tumor cells share some triggers, modulators, and end-effectors of the intermittent episodes of ischemia and reperfusion that characterize ischemic preconditioning and postconditioning. These are among the most effective maneuvers protecting cells from ischemia-reperfusion injury. If this hypothesis were confirmed, several well-investigated molecular mediators of pre/post-conditioning could be explored as therapeutic targets against tumor malignancy. For examples, drugs that completely block the cardioprotection induced by ischemic preconditioning, such as mitochondrial potassium ATP channel inhibitors or mitochondrial permeability transition pore openers, could be extraordinarily efficient in counteracting the adaptations of tumor cells and cancer stem cells to intermittent hypoxia. As a consequence, this strategy should be effective in blunting tumor capacity to progress toward malignancy and survive in ischemic conditions.

Suberanilohydroxamic acid prevents TGF-β1-induced COX-2 repression in human lung fibroblasts post-transcriptionally by TIA-1 downregulation

Cyclooxygenase-2 (COX-2), with its main antifibrotic metabolite PGE2, is regarded as an antifibrotic gene. Repressed COX-2 expression and deficient PGE2 have been shown to contribute to the activation of lung fibroblasts and excessive deposition of collagen in pulmonary fibrosis. We have previously demonstrated that COX-2 expression in lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) is epigenetically silenced and can be restored by epigenetic inhibitors. This study aimed to investigate whether COX-2 downregulation induced by the profibrotic cytokine transforming growth factor-β1 (TGF-β1) in normal lung fibroblasts could be prevented by epigenetic inhibitors. We found that COX-2 protein expression and PGE2 production were markedly reduced by TGF-β1 and this was prevented by the pan-histone deacetylase inhibitor suberanilohydroxamic acid (SAHA) and to a lesser extent by the DNA demethylating agent Decitabine (DAC), but not by the G9a histone methyltransferase (HMT) inhibitor BIX01294 or the EZH2 HMT inhibitor 3-deazaneplanocin A (DZNep). However, chromatin immunoprecipitation assay revealed that the effect of SAHA was unlikely mediated by histone modifications. Instead 3′-untranslated region (3′-UTR) luciferase reporter assay indicated the involvement of post-transcriptional mechanisms. This was supported by the downregulation by SAHA of the 3′-UTR mRNA binding protein TIA-1 (T-cell intracellular antigen-1), a negative regulator of COX-2 translation. Furthermore, TIA-1 knockdown by siRNA mimicked the effect of SAHA on COX-2 expression. These findings suggest SAHA can prevent TGF-β1-induced COX-2 repression in lung fibroblasts post-transcriptionally through a novel TIA-1-dependent mechanism and provide new insights into the mechanisms underlying its potential antifibrotic activity. Abbreviations Unlabelled Table SAHA suberanilohydroxamic acid TGF-β1 transforming growth factor-β1 COX-2 cyclooxygenase-2 TIA-1 T-cell intracellular antigen-1 PGE2 prostaglandin E2 IPF idiopathic pulmonary fibrosis DAC Decitabine HMT histone methyltransferase EZH2 enhancer of zeste homolog 2 DZNep 3-deazaneplanocin A 3′-UTR 3′-untranslated region α-SMA α-smooth muscle actin ECM extracellular matrix COL1 collagen 1 DNMT DNA methyltransferase HAT histone acetyltransferase HDAC histone deacetylase H3K9me3 histone H3 lysine 9 trimethylation ARE AUUUA-rich element HuR human antigen R ELAV1 ELAV-like RNA binding protein 1 TTP Tristetraprolin CUGBP2 CUG triplet repeat, RNA binding protein 2 F-NL fibroblast from non-fibrotic lung FCS fetal calf serum

Reliable measurement of E. coli single cell fluorescence distribution using a standard microscope set-up

BackgroundQuantifying gene expression at single cell level is fundamental for the complete characterization of synthetic gene circuits, due to the significant impact of noise and inter-cellular variability on the system’s functionality. Commercial set-ups that allow the acquisition of fluorescent signal at single cell level (flow cytometers or quantitative microscopes) are expensive apparatuses that are hardly affordable by small laboratories.MethodsA protocol that makes a standard optical microscope able to acquire quantitative, single cell, fluorescent data from a bacterial population transformed with synthetic gene circuitry is presented. Single cell fluorescence values, acquired with a microscope set-up and processed with custom-made software, are compared with results that were obtained with a flow cytometer in a bacterial population transformed with the same gene circuitry.ResultsThe high correlation between data from the two experimental set-ups, with a correlation coefficient computed over the tested dynamic range > 0.99, proves that a standard optical microscope– when coupled with appropriate software for image processing– might be used for quantitative single-cell fluorescence measurements. The calibration of the set-up, together with its validation, is described.ConclusionsThe experimental protocol described in this paper makes quantitative measurement of single cell fluorescence accessible to laboratories equipped with standard optical microscope set-ups. Our method allows for an affordable measurement/quantification of intercellular variability, whose better understanding of this phenomenon will improve our comprehension of cellular behaviors and the design of synthetic gene circuits. All the required software is freely available to the synthetic biology community (MUSIQ Microscope flUorescence SIngle cell Quantification).