Mimmo Turano

Increased HEXIM1 expression during erythroleukemia and neuroblastoma cell differentiation

The HEXIM1 protein, in association with 7SK snRNA, binds and inhibits the kinase activity of P‐TEFb (CDK9/cyclin T). P‐TEFb activity is crucial for efficient transcription elongation of viral and cellular genes. HEXIM1 was originally isolated as a protein up‐regulated by hexamethylene bisacetamide (HMBA), a prototypical inducer of differentiation. To determine the causative role of HEXIM1 during cell differentiation we analyzed the biochemical and functional consequences of HEXIM1 protein levels in several in vitro differentiation systems. We found that HEXIM1 mRNA and protein levels are up‐regulated during differentiation of murine erythroleukemia cells upon treatment with HMBA or DMSO. Stimulation of HEXIM1 is not restricted to hematopoietic cells, as induction of phenotypic differentiation of neuroblastoma cells by retinoic acid results in up‐regulation of HEXIM1. Moreover, ectopic expression of HEXIM1 causes growth inhibition and promotes neuronal differentiation. These findings highlight a crucial role of HEXIM1 protein during cell differentiation.

Human dyskerin: beyond telomeres

Abstract Human dyskerin is an evolutively conserved protein that participates in diverse nuclear complexes: the H/ACA snoRNPs, that control ribosome biogenesis, RNA pseudouridylation, and stability of H/ACA snoRNAs; the scaRNPs, that control pseudouridylation of snRNAs; and the telomerase active holoenzyme, which safeguards telomere integrity. The biological importance of dyskerin is further outlined by the fact that its deficiency causes the X-linked dyskeratosis congenita disease, while its over-expression characterizes several types of cancers and has been proposed as prognostic marker. The role of dyskerin in telomere maintenance has widely been discussed, while its functions as H/ACA sno/scaRNP component has been so far mostly overlooked and represent the main goal of this review. Here we summarize how increasing evidence indicates that the snoRNA/microRNA pathways can be interlaced, and that dyskerin-dependent RNA pseudouridylation represents a flexible mechanism able to modulate RNA function in different ways, including modulation of splicing, change of mRNA coding properties, and selective regulation of IRES-dependent translation. We also propose a speculative model that suggests that the dynamics of pre-assembly and nuclear import of H/ACA RNPs are crucial regulatory steps that can be finely controlled in the cytoplasm in response to developmental, differentiative and stress stimuli.

Real-time PCR quantification of human DKC1 expression in colorectal cancer

inary Tract Cancer Cooperative Group. J Clin Oncol 1997;/ 15:/1837 43. [7] Krarup-Hansen A, Helweg-Larsen S, Schmalbruch H, Rorth M, Krarup-C. Neuronal involvement in cisplatin neuropathy: Prospective clinical and neurophysiological studies. Brain 2007;/130:/1076 88. [8] Vigliani MC, Magistrello M, Polo P, Mutani R, Chio A. Risk of cancer in patients with Guillain-Barré syndrome (GBS). A population-based study. J Neurol 2004;/251:/321 6. [9] Tho LM, O’Leary CP, Horrocks I, Al-Ani A, Reeds NS. Guillain-Barre syndrome occurring after adjuvant chemoradiotherapy for endometrial cancer. Gynecol Oncol 2006;/ 100:/615 7. [10] Christodoulou C, Anastasopoulos D, Visvikis A, Mellan S, Detski I, Tsiakalos G, et al. Guillain-Barré syndrome in a patient with metastatic colon cancer receiving oxaliplatinbased chemotherapy. Anti-Cancer Drugs 2004;/15:/997 9.

Energy independent uptake and release of polystyrene nanoparticles in primary mammalian cell cultures

Nanoparticle (NPs) delivery systems in vivo promises to overcome many obstacles associated with the administration of drugs, vaccines, plasmid DNA and RNA materials, making the study of their cellular uptake a central issue in nanomedicine. The uptake of NPs may be influenced by the cell culture stage and the NPs physical-chemical properties. So far, controversial data on NPs uptake have been derived owing to the heterogeneity of NPs and the general use of immortalized cancer cell lines that often behave differently from each other and from primary mammalian cell cultures. Main aims of the present study were to investigate the uptake, endocytosis pathways, intracellular fate and release of well standardized model particles, i.e. fluorescent 44 nm polystyrene NPs (PS-NPs), on two primary mammalian cell cultures, i.e. bovine oviductal epithelial cells (BOEC) and human colon fibroblasts (HCF) by confocal microscopy and spectrofluorimetric analysis. Different drugs and conditions that inhibit specific internalization routes were used to understand the mechanisms that mediate PS-NP uptake. Our data showed that PS-NPs are rapidly internalized by both cell types 1) with similar saturation kinetics; 2) through ATP-independent processes, and 3) quickly released in the culture medium. Our results suggest that PS-NPs are able to rapidly cross the cell membrane through passive translocation during both uptake and release, and emphasize the need to carefully design NPs for drug delivery, to ensure their selective uptake and to optimize their retainment in the targeted cells.

A new human dyskerin isoform with cytoplasmic localization

BACKGROUND The human DKC1 gene is causative of X-linked dyskeratosis congenita (X-DC), a syndrome characterized by mucocutaneous features, bone marrow failure, tumor susceptibility, perturbation of stem cell function, and premature aging. DKC1 is thought to produce a single protein, named dyskerin, which shows strict nucleolar localization and participates in at least two distinct nuclear functional complexes: the H/ACA small nucleolar ribonucleoproteic complex involved in RNA pseudouridylation and the active telomerase complex. METHODS By bioinformatics and molecular analyses we identified a DKC1 splice variant able to encode a truncated form of dyskerin, confirmed its active expression in diverse human tissues by RT-PCR, and showed by immunoblotting and immunocytochemistry experiments that it actually encodes a novel protein. Stably transfected clones over-expressing the new isoform were analyzed for growth, morphology and adhesion properties. RESULTS Our results show that DKC1 encodes a new alternatively spliced mRNA able to direct the synthesis of a variant dyskerin with unexpected cytoplasmic localization. Intriguingly, when over-expressed in HeLa cells, the new isoform promotes cell to cell and cell to substratum adhesion, increases the cell proliferation rate and leads to cytokeratin hyper-expression. CONCLUSIONS AND GENERAL SIGNIFICANCE Our results highlight a novel degree of complexity and regulation of the human DKC1 gene and reveal that it can play a further, unpredicted role in cell adhesion. The identification of a dyskerin cytoplasmic variant reinforces the view that other mechanisms, in addition to telomere instability, can significantly contribute to the pathogenesis of the X-DC, and suggests that DKC1 nucleolar and cytoplasmic functions might cumulatively account for the plethora of manifestations displayed by this syndrome.

A novel Drosophila antisense scaRNA with a predicted guide function

A significant portion of eukaryotic small ncRNA transcriptome is composed by small nucleolar RNAs. From archaeal to mammalian cells, these molecules act as guides in the site-specific pseudouridylation or methylation of target RNAs. We used a bioinformatics search program to detect Drosophila putative orthologues of U79, one out of ten snoRNAs produced by GAS5, a human ncRNA involved in apoptosis, susceptibility to cancer and autoimmune diseases. This search led to the definition of a list of U79-related fly snoRNAs whose genomic organization, evolution and expression strategy are discussed here. We report that an intriguing novel specimen, named Dm46E3, is transcribed as a longer, unspliced precursor from the reverse strand of eiger, a fly regulatory gene that plays a key role in cell differentiation, apoptosis and immune response. Expression of Dm46E3 was found significantly up-regulated in a mutant strain in which eiger transcription is greatly reduced, suggesting that these two sense-antisense genes may be mutually regulated. Relevant to its function, Dm46E3 concentrated specifically in the Cajal bodies, followed a dynamic spatial expression profile during embryogenesis and displayed a degenerate antisense element that enables it to target U1b, a developmentally regulated isoform of the U1 spliceosomal snRNA that is particularly abundant in embryos.

A functional connection between dyskerin and energy metabolism

The human DKC1 gene encodes dyskerin, an evolutionarily conserved nuclear protein whose overexpression represents a common trait of many types of aggressive sporadic cancers. As a crucial component of the nuclear H/ACA snoRNP complexes, dyskerin is involved in a variety of essential processes, including telomere maintenance, splicing efficiency, ribosome biogenesis, snoRNAs stabilization and stress response. Although multiple minor dyskerin splicing isoforms have been identified, their functions remain to be defined. Considering that low-abundance splice variants could contribute to the wide functional repertoire attributed to dyskerin, possibly having more specialized tasks or playing significant roles in changing cell status, we investigated in more detail the biological roles of a truncated dyskerin isoform that lacks the C-terminal nuclear localization signal and shows a prevalent cytoplasmic localization. Here we show that this dyskerin variant can boost energy metabolism and improve respiration, ultimately conferring a ROS adaptive response and a growth advantage to cells. These results reveal an unexpected involvement of DKC1 in energy metabolism, highlighting a previously underscored role in the regulation of metabolic cell homeostasis.

Specific Effects of Chronic Dietary Exposure to Chlorpyrifos on Brain Gene Expression—A Mouse Study

Chlorpyrifos (CPF) is an organophosphate insecticide used to control pests on a variety of food and feed crops. In mammals, maternal exposure to CPF has been reported to induce cerebral cortex thinning, alteration of long-term brain cognitive function, and Parkinson-like symptoms, but the mechanisms of these processes are not fully understood. In this study, we aimed to gain a deeper understanding of the alterations induced in the brains of mice chronically exposed to CPF by dietary intake. For our purpose, we analysed F1 offspring (sacrificed at 3 and 8 months) of Mus musculus, treated in utero and postnatally with 3 different doses of CPF (0.1-1-10 mg/kg/day). Using RT2 Profiler PCR Arrays, we evaluated the alterations in the expression of 84 genes associated with neurodegenerative diseases. In the brains of exposed mice, we evidenced a clear dose–response relationship for AChE inhibition and alterations of gene expression. Some of the genes that were steadily down-regulated, such as Pink1, Park 2, Sv2b, Gabbr2, Sept5 and Atxn2, were directly related to Parkinson’s onset. Our experimental results shed light on the possibility that long-term CPF exposure may exert membrane signalling alterations which make brain cells more susceptible to develop neurodegenerative diseases.

Anatomy, photochemical activity, and DNA polymorphism in leaves of dwarf tomato irradiated with X-rays

The response of higher plants to ionising radiation depends on factors related to both radiation properties and plant features including species, cultivar, age, and structural complexity of the target organ. Adult plants of dwarf tomato were irradiated with different doses of X-rays to investigate possible variations in leaf morpho-anatomical traits, photosynthetic efficiency, and genomic DNA. In order to assess if and how responses depend on leaf developmental stage, we analysed two types of leaves; nearly mature leaves (L1) and actively developing leaves (L2), whose lamina size corresponded to 70 and 25 %, respectively, of the lamina size of the fully expanded leaves. The results show that the X-rays prevented full lamina expansion of the L2 leaves at all doses and induced early death of tissue of plants irradiated with doses higher than 20 Gy. Most anatomical modifications were not clearly dose-dependent and the radiation-induced increase in phenolic compounds was irrespective of dose. At high doses of X-rays (50 and 100 Gy), photochemical efficiency decreased significantly in both leaf types, whereas total chlorophyll content significantly decreased only in the L2 leaves. The random amplification of polymorphic DNA data show that the X-rays induced mutagenic effects in the L2 leaves even at low doses despite the absence of severe phenotypic alterations. Genetic structure found in the population of samples corroborates the results of anatomical and eco-physiological analyses: the 20 Gy dose seems to mark the threshold dose above which genetic alterations, structural anomalies, and perturbations in the photosynthetic apparatus become significant, especially in the actively expanding leaves.

Characterisation of mesenchymal colon tumour-derived cells in tumourspheres as a model for colorectal cancer progression

Cellular plasticity, the ability of cells to switch from an epitheial phenotype to a mesenchymal one and vice versa, plays a crucial role in tumour progression and metastases development. In 20-25% of patients with colon cancer and in 18% of patients with rectal cancer, metastases are present at the time of the first diagnosis. They are the first cause of colorectal cancer (CRC)-related mortality, defining stage IV CRC, which is characterized by a relatively short overall survival. We previously isolated two primary colon adenocarcinoma cell cultures that had undergone epithelial-mesenchymal transition (EMT), one with a high microsatellite instability phenotype (T88) and one with a chromosomal instability phenotype (T93). The aim of this study was to establish a model with which to study EMT, stemness features and cell plasticity in cancer progression and to examine the effects of incubation with lithium chloride (LiCl), a specific glycogen synthase kinase 3 β (GSK-3β) inhibitor, on these cellular processes. Indeed, GSK3β is an important regulator of cell survival, which promotes tumourigenesis in colon cells by facilitating the crosstalk between colorectal cancer pathways. Thus, we further characterized our system of adherent primary mesenchymal colon cancer cells and their paired tumourspheres by examining the expression and localisation of a panel of markers, including E- and N-cadherin, CD133, CD44v6, aldehyde dehydrogenase 1 (ALDH1) and leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5). We also characterised the molecular features of these tumourspheres and examined their response to LiCl. Furthermore, we explored the effects of LiCl on cell motility and plasticity. We demonstrated that LiCl reduced cell migration, stemness features and cell plasticity. We also observed the atypical nuclear localisation of membrane proteins, including N-cadherin, CD133 and CD44v6 in mesenchymal tumour cells. Of note, CD133 and CD44v6 appeared to localise at the plasma membrane in cells with a more epithelial phenotype, suggesting that the cytoplasmic/nuclear localisation of these proteins could favour and characterize cell plasticity in colorectal cancer progression.