Acrylamide: a probable catalytic topoisomerase II inhibitor

Rheumatoid arthritis-associated HLA-DRB1 genotypes in western Sicily

Metilazione del DNA in artrite reumatoide

Lo stato di metilazione del DNA genomico e del gene PTHrP è stato valutato con tecniche molecolari e citogenetiche in artrite reumatoide (AR), patologia autoimmune caratterizzata anche da alta incidenza di linfomi e da ipercalcemia per overespressione del gene PTHrP. La metilazione del DNA, infatti, ha un ruolo critico nello sviluppo delle malattie neoplastiche; il gene PTHrP avendo tre promotori uno dei quali contiene un’isola CpG è un buon candidato per la deregolazione da alterato pattern di metilazione locale. Le indagini sulla metilazione genomica, condotte su DNA estratto da sangue periferico di pazienti e di donatori e amplificato in reazioni di Methylation-Sensitive Arbitrarily Prim…

Luci ed ombre sul DNA: non è solo un modo di dire!

E' ormai assodato che ogni essere vivente, uomo compreso, ha caratteristiche che meglio lo fanno adattare all'ambiente in cui vive ed è anche altrettanto noto che all'interno di una stessa popolazione di viventi la presenza di variabilità è garanzia di adattamento all'ambiente mutevole. La variabilità genetica ha origine nel DNA, unica macromolecola in grado di gestire identità e diversità dell'informazione al susseguirsi delle generazioni; questo, nel grande tempo evoluzionistico, attraverso meccanismi di mutazione genetica e selezione ambientale, ha fatto si che ogni singolo vivente erediti una capacità differenziale di adattarsi finemente all'ambiente in cui vive. Per una cellula umana, …

Chromosomal abnormalities in Waldenström's macroglobulinemia

We report the results of cytogenetic studies of direct bone marrow (BM) preparations and of short-term BM and peripheral blood (PB) cultures from 17 patients with Waldenström's macroglobulinemia. We noted clonal chromosome changes in 10 patients. Abnormalities affected chromosomes X, Y, 2, 4, 5, 15, 16, 18, 19, 20, 21, and 22; in particular, chromosomes 2, 4, and 5 were involved in structural changes: a homogeneously staining region [hsr(2)], a der(4)t(4;?)(q32;?), and a 5q+. The other chromosomes were involved in numerical abnormalities, such as pseudodiploidy (a 46,X,-X,+15 clone), loss of chromosome Y, and monosomy of chromosomes 16, 18, 19, 20, 21, and 22. Nonclonal chromosome rearrange…

Telomerase activity in cells with arsenic-induced genomic instability

It is well known that the occurrence of dicentric chromosomes represent signature of telomere dysfunction and is a clear symptom of genomic instability. V79 Chinese hamster cells, treated with 10μM sodium arsenite for 24h and allowed to grow in drug-free medium (ASO cells), showed genomic instability with aneuploidy and nuclear abnormalities as well as the appearance of dicentric chromosomes since the 90th cell generation. TRAP assay was performed on growing ASO cells and on clones isolated during the course of the expanded growth. As expected, some clones with dicentric chromosomes and severely reduced telomerase activity went to death; surprisingly, other clones also bearing chromosomal e…

Possible involvement of the IL4 gene in Waldenström's macroglobulinemia

Abstract We report the results of a molecular investigation of 11 patients affected by Waldenstrom's macroglobulinemia, a rare B-cell malignancy characterized by an excessive proliferation of immunoglobulin(Ig)M-secreting plasmacytoid cells. In particular, we studied the interleukin-4 (IL4) gene, which codes for a B-specific growth factor capable of stimulating the proliferation and differentiation of secreting plasma cells. By Southern hybridization, in three patients we found the presence of additional bands in comparison with the expected pattern; moreover, these bands showed a different degree of intensity.

Acrilamide: un probabile inibitore della topoisomerasi II

Stato di metilazione del promotore 2 del gene PTHrP in pazienti affetti da mieloma multiplo

PTHrP in differentiating human mesenchymal stem cells: Transcript isoform expression, promoter methylation, and protein accumulation

Human PTHrP gene displays a complex organization with nine exons producing diverse mRNA variants due to alternative splicing at 5' and 3' ends and the existence of three different transcriptional promoters (P1, P2 and P3), two of which (P2 and P3) contain CpG islands. It is known that the expression of PTHrP isoforms may be differentially regulated in a developmental stage- and tissue-specific manner. To search for novel molecular markers of stemness/differentiation, here we have examined isoform expression in fat-derived mesenchymal stem cells both maintained in stem conditions and induced toward adipo- and osteogenesis. In addition, the expression of the splicing isoforms derived from P2 …

Cytogenetic characterization of HB2 epithelial cells from the human breast.

HB2 is a cell line originated by subcloning of MTSV1-7 mammary luminal epithelial cells isolated from human milk and immortalization via introduction of the gene encoding simian virus 40 (SV40) large T antigen. Despite its wide utilization as non-neoplastic counterpart in assays aimed to elucidating various biochemical and genetical aspects of normal and tumoral breast cells, to our knowledge no literature data have so far appeared concerning the chromosomal characterization of the HB2 cells. Here, we report the cytogenetic characterization of the karyotype of HB2 cells, which puts in evidence the occurrence of changes in chromosomal number and structure and the presence of unidentified chr…

Genomic instability induced by α-pinene in Chinese hamster cell line.

Here, we report the effects of exposure of mammalian cells to α-pinene, a bicyclic monoterpene used in insecticides, solvents and perfumes. Morphological analysis, performed in V79-Cl3 cells exposed for 1 h to increasing concentrations (25 up to 50 μM) of α-pinene, indicated a statistically significant increase in micronucleated and multinucleated cell frequencies; apoptotic cells were seen at 40 and 50 μM. This monoterpene caused genomic instability by interfering with mitotic process; in fact, 50% of cells (versus 19% of control cells) showed irregular mitosis with multipolar or incorrectly localised spindles. Cytogenetic analysis demonstrated high-frequency hypodiploid metaphases as well…

Nutrigenetics, nutrigenomics and phenotypic outcomes of dietary low-dose alcohol consumption in the suppression and induction of cancer development: evidence from in vitro studies

It is known that the intake of alcoholic beverages may impair genetic and epigenetic regulatory events with consequent crucial effects on cell phenotypes and that its association with selected genotypes can lead to a different risk of cancer in the population. The aim of this review is to pick up selected studies on this topic and recapitulate some of the biochemical and nutrigenetic/nutrigenomic aspects involved in the impact of dietary low-dose alcohol consumption on the switching-on or -off of tumorigenic pathways. These include i) the existence of predisposing or protective human genotypes and the relationship between dietary compounds and alcohol in the promotion or inhibition of carci…

Arsenic-induced DNA hypomethylation affects chromosomal instability in mammalian cells

Early genetic instability induced in dividing V79-Cl3 Chinese hamster cells by inorganic arsenic, as demonstrated in our previous investigation, was evidenced by aneuploidy and nuclear abnormalities, but not by chromosomal rearrangements. Here we report the results of cytogenetic and morphological analyses performed on the progeny of cells dividing at the end of sodium arsenite treatment after they had been expanded through 120 generations (ASO cells) and then cloned. The acquired genetic instability persisted and was increased by highly unstable chromosomal rearrangements, namely dicentric chromosomes and telomeric associations, which were not seen following acute exposure. A peculiar find…

Role and importance of polymorphisms with respect to DNA methylation for the expression of CYP2E1 enzyme

Different individuals possess slightly different genetic information and show genetically-determined differences in several enzyme activities due to genetic variability. Following an integrated approach, we studied the polymorphisms and methylation of sites contained in the 5' flanking region of the metabolizing enzyme CYP2E1 in correlation to its expression in both tumor and non-neoplastic liver cell lines, since to date little is known about the influence of these (epi)genetic elements in basal conditions and under induction by the specific inductor and a demethylating agent. In treated cells, reduced DNA methylation, assessed both at genomic and gene level, was not consistently associate…

Role of “mate tea” extracts modulating DNA methylation: in vitro studies for future significances for human health

It is known that a healthy and balanced diet is essential for maintaining a state of good health of the individual, and that the onset of many diseases is related to inadequate nutrition. Many small molecules contained in foods (Food Small Molecules, SFMs) have the ability to influence the functioning of many cellular metabolic pathways. The term nutrigenomics, in fact, refers to an emerging branch of genetics and biochemistry which has the aim of identifying how the nutrients can determine the innate risk of developing diseases (diabetes, obesity, cardiovascular disease and some cancers). In addition, with the help of epigenetics, it is possible to further understand how the SFMs act on DN…

L'incontro tra la chimica, la biologia e le tecnologie

Un incontro, per essere proficuo, deve sempre generare qualcosa e questo vale per tutto! Quando le leggi della chimica, apparentemente astratte, si applicano alle biomolecole, permettendo la vita, hanno compiuto un primo proficuo incontro dal quale si è generata la chimica biologica. Se, successivamente, a quest’ultima si associano le possibilità tecnologiche inventate dall’uomo, che facilitando le procedure le rendono possibili e fruibili a molti, l’incontro diventa ancora più produttivo e si generano le biotecnologie. E’ grazie a queste tecnologie che è stato possibile il salto dalla genetica classica, chiamata diretta, a quella molecolare con più approfondita conoscenza del genoma umano …

Methylation of cytokines gene promoters in IL-1β-treated human intestinal epithelial cells

Objective and design: Epigenetic regulation is important in the activation of inflammatory cells. In the present study, we evaluated if DNA-methylation variations are involved in Interleukin-1β (IL-1β)-induced intestinal epithelial cells activation. Materials and methods: Differentiated Caco-2 cells were exposed to IL-1β or to 5-azadeoxycytidine (5-azadC) for 24 or 48 h. Genome-wide methylation status was evaluated, while DNA methylation status at the promoter region of the gene encoding interleukin-6, 8 and 10 (IL-6, 8 and 10) was estimated. The levels of the corresponding gene products as well as DNA methyltransferases (DNMTs) quantity were assessed. Results: IL-1β decreased genomic m…

A new pericentric inv(8) in acute nonlymphocytic leukemia.

Abstract Chromosome studies were carried out on unstimulated bone marrow cells from a patient with a diagnosis of acute nonlymphocytic leukemia (FAB M6 ANLL). Cytogenetic analysis revealed a mosaic chromosome pattern 46,XX46,XX,inv(8)(p21q24). This pericentric inversion has not been previously described in ANLL. Because, fragile sites, zinc finger gene loci, and the MYC protooncogene have been localized to band 8q24, a putative role for these sites and genes could be considered.

Pro-apoptotic activity of the phytochemical Indicaxanthin on colorectal carcinoma cells (Caco-2) and epigenetic CpG demethylation of the promoter and reactivation of the expression of p16

Phytochemicals play prominent roles in human diet and nutrition as protective redo-active substances in prevention of several disorders and chronic diseases in humans. Today, their function as potent modulators of the mammalian epigenome-regulated gene expression is rapidly emerging. In the present study antiproliferative effects of Indicaxanthin (Ind) from the fruits of Opuntia ficus-indica (1), and potential influence on DNA methylation has been investigated on Caco-2 cells, a human cell line of colorectal carcinoma. Ind caused a clear dose- and time-dependent decrease of the cell proliferation (IC(50) 50 M) associated to apoptosis as demonstrated by phosphatidylserine externalization an…

The expression of PTHrP isoforms in differentiating human fat-derived mesenchymal stem cells

UPREGULATION OF MIR-29A AND GENOMIC DNA HYPERMETHYLATION IN NORMAL KARYOTYPE AML SHOWING DNMT3A MUTATION UPREGOLAZIONE DEL MIR-29A E IPERMETILAZIONE DEL DNA GENOMICO NELLE LAM A CARIOTIPO NORMALE DNMT3A MUTATE

DNMT3A, a member of DNA methyltransferases, is mutated in approximately 22% of de novo normal karyotype acute myeloid leukemia (NK-AML) patients leading to adverse overall survival. The highly recurrent mutation in DNMT3A is a “gain of function-like” at codon R882. To indagate about miRNA signature in NK-AML R882-DNMT3A mutated we studied by qRT-PCR the expression of 384 known human miRNA in 9 selected de-novo AML DNMT3A mutated. We compared miRNA expression data with our previous results obtained in 31 AML DNMT3A wild type (WT) and we focused on a strong up-regulation of miR155, miR29a, miR196b and miR25. We consolidated this data in additional 24 new DNMT3A mutated AML and we confirmed th…

Biological effects of inorganic arsenic on primary cultures of rat astrocytes

It is well established that inorganic arsenic induces neurotoxic effects and neurological defects in humans and laboratory animals. The cellular and molecular mechanisms of its actions, however, remain elusive. Herein we report the effects of arsenite (NaAsO2) on primary cultures of rat astrocytes. Cells underwent induction of heat shock protein 70 only at the highest doses of inorganic arsenic (30 and 60 microM), suggesting a high threshold to respond to stress. We also investigated arsenic genotoxicity with the comet assay. Interestingly, although cells treated with 10 microM arsenite for 24 h maintained >70% viability, with respect to untreated cells, high DNA damage was already observed…

Antagonist effects of Acrylamide on clastogenity of VP16

We investigated on the Acrylamide (AA) capability of influencing the clastogenic effects of VP16, the topoisomerase II targeting drug, by performing sequential treatments in V79 Chinese hamster cells. The VP16 cytotoxicity resulted almost completely antagonized by preincubating cells with nontoxic concentrations of AA, as inferred by statistically significant differences versus response with VP16 alone. Moreover, the severe clastogenic effect of VP16, evidenced by the presence of complex structural chromosome aberrations and by high frequencies of micronulei and sister chromatid exchanges, was reduced by AA in a dose-dependent manner. For example, the frequency of micronucleated cells induc…

DNA-methylation dependent regulation of embryo-specific 5S ribosomal DNA cluster transcription in adult tissues of sea urchin Paracentrotus lividus

Abstract We have previously reported a molecular and cytogenetic characterization of three different 5S rDNA clusters in the sea urchin Paracentrotus lividus and recently, demonstrated the presence of high heterogeneity in functional 5S rRNA. In this paper, we show some important distinctive data on 5S rRNA transcription for this organism. Using single strand conformation polymorphism (SSCP) analysis, we demonstrate the existence of two classes of 5S rRNA, one which is embryo-specific and encoded by the smallest (700 bp) cluster and the other which is expressed at every stage and encoded by longer clusters (900 and 950 bp). We also demonstrate that the embryo-specific class of 5S rRNA is ex…

Genomewide hypomethylation and PTHrP gene hypermethylation as a model for the prediction of cancer risk in rheumatoid arthritis

We have previously shown that PTHrP(38-94)-amide restrains growth and invasion "in vitro", causes striking toxicity and accelerates death of some breast cancer cell lines, the most responsive being MDA-MB231 whose tumorigenesis was also attenuated "in vivo". PTHrP(38-94)-amide contains the domain implicated in the nuclear import of PTHrP. Although the nucleus was identified as a destination for mid-region PTHrP, evidence for direct DNA-binding capability is lacking to date. Here, we examined the localization of PTHrP(38-94)-amide within MDA-MB231 cells and within metaphase spread preparations and characterized its DNA-binding properties, employing a combination of immunocytochemical, cytoge…

La conservazione della biodiversità dei boschi nativi: un investimento in “salute genetica”

Tutti i viventi sono fenotipicamente diversi fra loro e mostrano, anche all’interno della stessa specie, variabilità fenotipica che li rende peculiari e unici. Soltanto in uno scenario di grande diversità, infatti, è possibile distinguere un individuo da un altro e di conseguenza può essere asserito che l’unicità è la funzione della diversità, tanto per gli animali che per i vegetali. Queste differenze fenotipiche sottintendono, com’è ovvio, una differenza genotipica, una variabilità genetica che è la base della biodiversità all’interno di uno stesso genere. Prendendo in considerazione il genere Prosopis, ad esempio, un albero giudicato patrimonio boschivo in Argentina, possiamo asserire ch…

Nutraceuticals and Functional Foods. Experimental research as a means for the innovation of already existing products: reporting of a type case.

Innovation and the creation of new products in a company, or in a food industry, is an expensive process and requires profound transformations of the production processes. For this reason, the governance of some businesses often gives up on the idea of innovating, especially if some of their products, driving the market, can ensure short and medium-term earnings security. It is also quite well known, however, that the long-term solidity of a company is ensured only by innovation: mediating between these two possibilities is very difficult but one sustainable possibility, today, may consist in functionalizing already existing food products, adding, in the industrial manufacturing process, bi…

Genetic polymorphism of the bitter taste TAS2R38 gene in central Sicily

Phytochemical Indicaxanthin Inhibits Colon Cancer Cell Growth and Affects the DNA Methylation Status by Influencing Epigenetically Modifying Enzyme Expression and Activity

<b><i>Background:</i></b> Recently, we have shown anti-proliferative and pro-apoptotic effects of indicaxanthin associated with epigenetic modulation of the onco-suppressor <i>p16</i><sup><i>INK4a</i></sup> in the human colon cancer cell line CACO2. In the present study, the epigenetic activity of indicaxanthin and the mechanisms involved were further investigated in other colorectal cancer cell lines. <b><i>Methods:</i></b> LOVO1, CACO2, HT29, HCT116, and DLD1 cells were used to evaluate the potential influence of consistent dietary concentrations of indicaxanthin on DNA methylation, and the epigenetic mech…

SNP variation in the bitter taste TAS2R38 gene evaluated in student populations of several italian universities and isolates

People vary widely in their sensitivities to bitter compounds, but the all intercorrelation of these sensitivities is unknown. The study of genetic influences on bitter taste perception originated from the discovery in the 1930s that some individuals had taste to phenylthiocarbamide(PTC), whereas others found it extremely bitter. Subsequently, many studies were carried out on PTC and the structurally related compound propylthiouracil (PROP) to assess this viability and to determine the root causes. Initial family studies strongly suggested that PTC no tasting was due to a recessive allele in a single gene and heritability was estimated at 0,5. 55-85% of variation in PTC detection. The PTC ge…

PTHrP [38-94]-amide is a DNA-binding factor: cytogenetic and molecular evidence and biological effect on normal and neoplastic human breast cells

CR1 allelic distribution in healthy italians and SLE patients

Complement receptor type 1 (CR1; CD35), the receptor for C3b/C4b, has a molecular weight polymorphism involving four co-dominantly expressed alleles CR1*1, CR1*2, CR1*3 and CR1*4. The most common allotype in the Caucasian population is CR1*1. Previous studies on the frequency of the CR1 allele in Systemic Lupus Erythematosus (SLE) patients reported conflicting results. Recently an increased frequency of the CR1*2 allele was reported in Caucasian SLE patients. The aim of our study is to assess the frequency of CR1 structural alleles in a sample of the Italian population and in a cohort of Italian SLE patients equally distributed nationwide. We found that the most common phenotype was CR1*1.1…

Methylation decrease of BECN1 gene induced by phytochemical Indicaxantin in Caco2 cells: an epigenetic hypothesis of autophagy

Autophagy is a highly conserved catabolic process that degrades and recycles intracellular components through the lysosomes [1]. The role of this process in tumorigenesis and tumor progression is controversial: in the early stages, it can block tumor growth and conversely it can promote its progression in the later stages [2]. The tumor suppressor BECN1 gene, encodes the protein Beclin 1, a marker of autophagy down-regulated in several types of cancer, such as colorectal cancer [3]. There are a lot of both genetic and environmental risk factors for colorectal cancer, including diet: for this reason, in accordance with epidemiological studies, consumption of foods rich in phytochemicals is w…

The Histone Deacetylase Inhibitor JAHA Down-Regulates pERK and Global DNA Methylation in MDA-MB231 Breast Cancer Cells

The histone deacetylase inhibitor N-1-(ferrocenyl)-N-8-hydroxyoctanediamide (JAHA) down-regulates extracellular-signal-regulated kinase (ERK) and its activated form in triple-negative MDA-MB231 breast cancer cells after 18 h and up to 30 h of treatment, and to a lesser extent AKT and phospho-AKT after 30 h and up to 48 h of treatment. Also, DNA methyltransferase 1 (DNMT1), 3b and, to a lesser extent, 3a, downstream ERK targets, were down-regulated already at 18 h with an increase up to 48 h of exposure. Methylation-sensitive restriction arbitrarily-primed (MeSAP) polymerase chain reaction (PCR) analysis confirmed the ability of JAHA to induce genome-wide DNA hypomethylation at 48 h of expos…

Il progetto Mentori per la Didattica dell’Università degli Studi di Palermo: l’estensione del numero dei partecipanti, la figura del Mentore Senior e le esperienze di innovazione didattica

L’esperienza del Progetto Mentore (PM) nasce nel 2013 dalla percezione, da parte di diversi docenti dell’Ateneo di Palermo, che l’Accademia italiana stesse abbandonando a sé stessa la funzione didattica, confidando di fatto nelle capacità di ognuno di organizzarsi, aggiornarsi e valutarsi (Felisatti, Scialdone, Cannarozzo, & Pennisi, 2019; Caradonna, Morale, Pace, Scargiali, Scialdone, & Auteri, 2020). Tale impressione era tra le altre cose basata sul fatto che nessuno dei sistemi di valutazione dei docenti universitari (il processo di abilitazione scientifica nazionale, ma anche la stessa valutazione della qualità dei corsi di studio) mettesse enfasi sulla necessità da parte del do…

Persistent genomic instability by arsenic exposure in V79 Chinese hamster cells

Previously, we demonstrated that acute treatment with arsenic leads mammalian cells to exhibit persistent chromosomal instability and DNA hypomethylation, by performing investigations after about 2 months of subculturing. In order to evaluate quantitatively the continuing instability during the expanded growth, we carried out cytogenetic, morphologic and molecular analyses immediately after exposure and every week up to 112 cell generations. Briefly, V79 Chinese hamster cells were treated with 10 µM sodium arsenite (SA) for 24h; at the end of exposure, mitotic rounded-up cells were harvested by shake-off and allowed to grow in drug-free medium. The instability markers, micronucleated and mu…

Il Progetto “Mentori per la didattica” e le altre iniziative dell’Università di Palermo

L’Ateneo di Palermo sta portando avanti diverse iniziative di miglioramento della qualità della didattica. In particolare, il progetto “Mentori per la didattica” nasce nel 2013 su iniziativa di alcuni docenti di ingegneria con l’obiettivo di migliorare la qualità della didattica dei partecipanti grazie a due strumenti chiave: l’aiuto dei mentori e una serie di incontri di riflessione e approfondimento. Ogni docente (mentee) che aderisce al progetto ha due mentori che hanno il compito di aiutarlo a migliorare la qualità della sua didattica. Inoltre, egli stesso svolge il ruolo di mentore a favore di altri due colleghi, cosicché ciascuno è mentore e mentee allo stesso tempo. I mentori assisto…

IL-1β maintains the DNA hypermethylation of anti-inflammatory IL-10 gene in a human intestinal epithelial cell line

Intestinal inflammation is a natural process crucial to maintain gut integrity, but its deregulation is involved in the pathogenesis of severe intestinal disorders[1]. Intestinal epithelial cells play a crucial role in the inflammatory response, modulating the immune cell exposure to antigens and by their ability to secrete many inflammatory mediators. IL-1β represents a pivotal player: secreted by infiltrated leucocytes, it induces the expression of several pro-inflammatory genes. Also the anti-inflammatory IL-10, whose function is to terminate the inflammatory process, modulates the intestinal physiology[2]. Recent clinical reports showed that patients with ulcerative colitis in remission…

Non-flavonoid polyphenols in osteoporosis: preclinical evidence

The development of progressive osteopenia and osteoporosis (OP) is due to the imbalance between bone resorption and bone formation, determining a lower bone resistance, major risks of fractures, with consequent pain and functional limitations. Flavonoids, a class of polyphenols, have been extensively studied for their therapeutic activities against bone resorption, but less attention has been given to a whole series of molecules belonging to the polyphenolic compounds. However, these classes have begun to be studied for the treatment of OP. In this systematic review, comprehensive information is provided on non-flavonoid polyphenolic compounds, and we highlight pathways implicated in the ac…

Acrylamide catalytically inhibits topoisomerase II in V79 cells.

The vinyl monomer acrylamide is characterized by the presence of an alpha,beta-unsaturated carbonyl group that makes it reactive towards thiol, hydroxyl or amino groups and towards the nucleophilic centers in DNA. The ability of acrylamide to chemically modify protein thiols has prompted us to consider topoisomerase II as one possible target of acrylamide, since agents targeting protein sulfhydryl groups act as either catalytic inhibitors or poisons of topoisomerase II. Nuclear extracts from V79 Chinese hamster cells incubated with acrylamide reduced topoisomerase II activity as inferred by an inability to convert kinetoplast DNA to the decatenated form. Nuclear extracts incubated with acry…

The Binomial “Inflammation-Epigenetics” in Breast Cancer Progression and Bone Metastasis: IL-1β Actions Are Influenced by TET Inhibitor in MCF-7 Cell Line

The existence of a tight relationship between inflammation and epigenetics that in primary breast tumor cells can lead to tumor progression and the formation of bone metastases was investigated. It was highlighted how the induction of tumor progression and bone metastasis by Interleukin-1 beta, in a non-metastatic breast cancer cell line, MCF-7, was dependent on the de-methylating actions of ten-eleven translocation proteins (TETs). In fact, the inhibition of their activity by the Bobcat339 molecule, an inhibitor of TET enzymes, determined on the one hand, the modulation of the epithelial-mesenchymal transition process, and on the other hand, the reduction in the expression of markers of bo…

Nucleoplasmic bridges and acrocentric chromosome associations as early markers of exposure to low levels of ionising radiation in occupationally exposed hospital workers

Ionising radiation, with the contribution of telomere shortening, induces DNA double-strand breaks that result in chromosome end fusion, nucleoplasmic bridges (NPBs) and chromosome aberrations (ChAbs) as well as dicentric chromosomes. In order to investigate the chromosomal damage induced by occupational ionising radiation at low exposure levels, and to find early markers of health hazard, peripheral lymphocytes of occupationally exposed hospital workers were cytogenetically analysed. Results showed a significant difference in the frequency of ChAbs in exposed subjects relative to controls. A significant number of NPBs between nuclei of binucleated cultured lymphocytes from exposed subjects…

Parathyroid Hormone Related Protein (PTHrP)-Associated Molecular Signatures in Tissue Differentiation and Non-Tumoral Diseases

Bitter taste genetics and food preference in italian population

Objective: To investigate the possible role of the polymorphic bitter taste gene, TAS2R38, known to be involved in the perception of the bitter synthetic chemical phenylthiocarbamide (PTC), in influencing food preference and body mass index(BMI). Methods: up to now more than 1500 university students (17-25 years old) at Catania, Cosenza, Rome, Palermo, Pisa, Parma, Chieti, Trento University have been enrolled in the study. DNA was extracted from saliva, and genotyped by TaqMan assay for the most frequent polymorphism (PAV/AVI) of TAS2R38 gene. A possible association between genotype and food preference was assessed by administering a detailed questionnaire for food preferences and life styl…

Jay Amin Hydroxamic Acid (JAHA), a histone deacetylase inhibitor with cytotoxic activity and the property to increase DNA repair of triple-negative MDA-MB231 breast cancer cells.

Jay Amin Hydroxamic Acid (JAHA; N8-ferrocenylN1-hydroxy-octanediamide) is a ferrocene-containing analogue of the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA). JAHA’s cytotoxic activity on MDA-MB231 triple negative breast cancer (TNBC) cells at 72 h has been previously demonstrated with an IC50 of 8.45 M. JAHA’s lethal effect was found linked to perturbations of cell cycle, mitochondrial activity, signal transduction and autophagy mechanisms. In order to glean novel insights on how MDA-MB231 breast cancer cells respond to the cytotoxic effect induced by JAHA, and to compare the biological effect with the related compound SAHA, we have employed a combination o…

Origine ed evoluzione dei genomi: dal brodo primordiale al DNA umano

Per una migliore comprensione dell’origine e dell’evoluzione dei genomi è conveniente posizionare l’inizio e il decorso di tale processo all’interno della successione di evoluzioni che partono dall’origine dell’universo e che ancora oggi non si possono, per definizione, ritenere conclusi. Il Big Bang conclude l’evoluzione cosmica universale e dà origine all’evoluzione planetaria con la formazione dei sistemi solari ed i loro pianeti. Considerando il nostro sistema solare, e la terra in particolare, l’evoluzione geotermica, il raffreddamento del pianeta, fornisce una condizione fisica opportuna ad un’evoluzione chimica che, a conclusione, porta al brodo primordiale. Le molecole semplici poss…

Biological effects of alpha-pinene in cultured mammalian cells

In this work we report the effects of exposure of mammalian cells to α-pinene, a bicyclic monoterpene founded in essential oils and used in insecticides, solvents, perfumes, etc.. Morphological analysis, performed in V79 cells exposed to increasing doses(25μM up to 50μM) of α-pinene, indicated a increase of dose-related nuclear abnormalities; apoptotic cells were seen at higher doses. Immunofluorescence with anti β- tubulin antibody showed that monoterpene induced genomic instability by interfering with mitotic process; in fact, 50% (vs 19% in the control cells) of irregular mitosis with multipolar or not correctly localized spindles were detected, suggesting that α-pinene affects cell stab…

Arsenite-induced aneuploidy following short and long-term exposure in mammalian cells

We studied the long-term progression of chromosomal instability in V79 cells treated acutely with arsenite (10mM, 24 hr) followed by growth in arsenic-free medium for 120 cell generations. Indirect immunostaining using anti-ß-tubulin antibody showed severe alterations in spindle morphology after only 6 h treatment and cytogenetic investigations carried out at the end of treatment revealed that the percentage of cells with 21 chromosomes (modal number of the cell line) decreased, making way for aneuploid cells. The acquired instability remained and propagated within the cell population. Moreover, we treated V79-derived G12 cells with sub-lethal doses (0.1-1.0 μM) of arsenite for 10 days foll…

Science and Healthy Meals in the World: Nutritional Epigenomics and Nutrigenetics of the Mediterranean Diet

The Mediterranean Diet (MD), UNESCO Intangible Cultural Heritage of Humanity, has become a scientific topic of high interest due to its health benefits. The aim of this review is to pick up selected studies that report nutrigenomic or nutrigenetic data and recapitulate some of the biochemical/genomic/genetic aspects involved in the positive health effects of the MD. These include (i) the antioxidative potential of its constituents with protective effects against several diseases; (ii) the epigenetic and epigenomic effects exerted by food components, such as Indacaxanthin, Sulforaphane, and 3-Hydroxytyrosol among others, and their involvement in the modulation of miRNA expression; (iii) the …

Two immortalized rat astrocyte cell lines as in vitro model for specific cell proliferation studies: cytogenetic and epigenomic characterization and diversification

Here we report differences between: 1) a heterogeneous population of primary rat brain astrocytes (Primary), in culture since several years ago, and 2) a cloned cell line (Clone), obtained from the Primary cells. Both populations maintain astrocyte morphology but, according to cytogenetic and epigenomic characterization, differ for the chromosomal asset from rat normal cells (42 chromosomes): Primary cells show mostly a bimodal karyotype with 41 or 43 chromosomes, and Clone has a unique-modal karyotype of 43 chromosomes. Interestingly, we also found that both cell lines show genome-wide DNA hypomethylation, with Clone showing even more pronounced demethylation respect to Primary cells. Thes…

Genetic polymorphisms and epigenetics changes in human metabolizing enzymes genes to predict differential therapeutic drug effects

It has been understood that genetic variability can influence individual ability to metabolize drugs (Kiyohara C. et al., 2002). In particular, sequence changes into some genes give to subject a variable capability to response to a therapy protocol, to begin a resistance toward therapeutic drugs or, on the contrary, to be more sensible to it: the genes of CYP-family, CYP2A6 and CYP2E1, are good examples. Nevertheless, gene expression can be affected either by DNA sequence mutations (polymorphisms) or by “epigenetic modifications”, such as DNA methylation of a CpG islands in a gene promoter ion (Zhu J. et al., 2009). For these reasons, it is indispensable, today, to integrate genetic analyse…

Abnormal mitotic spindle assembly and cytokinesis induced by D-Limonene in cultured mammalian cells

D-Limonene is found widely in citrus and many other plant species; it is a major constituent of many essential oils and is used as a solvent for commercial purposes. With the discovery of its chemotherapeutic properties against cancer, it is important to investigate the biological effects of the exposure to D-Limonene and elucidate its, as yet unknown, mechanism of action. We reported here that D-Limonene is toxic in V79 Chinese hamster cells in a dose-dependent manner. Moreover, to determine the cellular target of D-Limonene, we performed morphological observations and immunocytochemical analysis and we showed that this drug has a direct effect on dividing cells preventing assembly of mito…

Esempi di regolazione del metabolismo cellulare e dell'organismo umano

Quanti sprechi ci sarebbero in una famiglia se non si riuscisse a bilanciare l’approvvigionamento di cibo deperibile con il suo consumo, prima della scadenza? E a quanti spiacevoli inconvenienti andremmo incontro se non si riuscisse a ben coordinare il prelievo di scorte alimentari, conservate in dispensa, con il loro ripristino, facendo la spesa? Sono due interrogativi che possiamo ben comprendere in quanto facenti parte della nostra vita quotidiana ma che ci aiutano a capire quale inevitabile compito deve essere costantemente mantenuto dentro una cellula, o, più faticosamente, in un organismo complesso, come l’uomo, ad esempio. La cellula in piccolo e l’organismo in grande sono consumator…

Characterization of three different clusters of 18S-26S ribosomal DNA genes in the sea urchin P. lividus: Genetic and epigenetic regulation synchronous to 5S rDNA.

We previously reported the characterization 5S ribosomal DNA (rDNA) clusters in the common sea urchin Paracentrotus lividus and demonstrated the presence of DNA methylation-dependent silencing of embryo specific 5S rDNA cluster in adult tissue. In this work, we show genetic and epigenetic characterization of 18S-26S rDNA clusters in this specie. The results indicate the presence of three different 18S-26S rDNA clusters with different Non-Transcribed Spacer (NTS) regions that have different chromosomal localizations. Moreover, we show that the two largest clusters are hyper-methylated in the promoter-containing NTS regions in adult tissues, as in the 5S rDNA. These findings demonstrate an an…

Acidi nucleici: struttura e funzione

Guardando il progetto di un tempio greco e quello di un moderno grattacielo, chiunque di noi trova dei tratti di similitudine e di divergenza, ma soprattutto scopre che il “linguaggio” usato per costruirlo è uguale: sia gli ingegneri di quei tempi sia quelli odierni hanno entrambi raffigurato in un progetto le fondamenta, le sopraelevazioni, i carichi da sostenere, le suddivisioni in stanze e altro. Ebbene, addentrandoci a studiare questo capitolo scopriremo che le informazioni su come generare e gestire un vivente sono contenute in un progetto fatto da macromolecole, gli acidi nucleici, scritte nel medesimo linguaggio, il codice genetico, uguale sin dai primi esseri viventi nati milioni di…

Cytochrome P450 2E1 variable number tandem repeat polymorphisms and health risks: A genotype-phenotype study in cancers associated with drinking and/or smoking

Cytochrome P450 2E1 (CYP2E1) is one of the main enzymes involved in the oxidation of ethanol and in the transformation of a number of potentially dangerous compounds. It has various polymorphic sites, one of which is a variable number tandem repeat (VNTR) polymorphism previously described in the 5'-flanking region. The aim of this study was to investigate the genotype-phenotype association between CYP2E1 VNTR polymorphisms and risky health habits in healthy subjects and to analyze the associations between these polymorphisms with drinking- and/or smoking-related cancers. We analyzed 166 healthy subjects by genotyping for the CYP2E1 VNTR polymorphism associated with drinking and/or smoking h…

Le metiltrasferasi (DNMT): macchine molecolari che regolano l'espressione genica attraverso meccanismi epigenetici

Oggi è noto che un gene si esprime non soltanto in base alla sequenza primaria del suo promotore, ma anche in base alla possibilità che la cromatina locale ha di accogliere fattori di trascrizione trans-agenti. La metilazione della citosina del DNA e uno fra i più studiati meccanismi di rimodellamento della cromatina e quindi di regolazione dell'espressione genica, maggiormente negli eucarioti. Spostare metili, metilare o demetilare opportune sequenze regolatorie dei geni significa regolare la loro espressione genica senza modificare la sequenza del DNA e questo è oggetto di studio dell'Epigenetica, una scienza di nuovo interesse, che riesce a spiegare fenomeni prima non a pieno compresi, i…

POLIMORFISMI DEI GENI CYP2A6 E CYP2E1 IN RELAZIONE A STILI DI VITA IN UNA POPOLAZIONE DELLA SICILIA CENTRO-OCCIDENTALE

Le più recenti stime dell’OMS testimoniano che le patologie correlate all’abitudine al fumo e al consumo di alcool sono tra le principali cause di morte nei soggetti adulti ed è noto che la diversità interindividuale nell’adottare tali stili di vita è attribuibile, oltre che a fattori psico-sociali, anche a polimorfismi dei geni per i citocromi P450. Questo studio mira a verificare se esista una correlazione fra i genotipi CYP2A6 -CYP2E1 e abitudine al fumo e consumo d’alcool nella Sicilia centro-occidentale. I risultati ottenuti dall’analisi della distribuzione di alcuni alleli di entrambi i geni in 65 soggetti (41 donne e 24 uomini) hanno evidenziato che i genotipi CYP2A6*1/*4A e CYP2A6*1…

Flavonoids in Bone Erosive Diseases: Perspectives in Osteoporosis Treatment

Imbalance of bone homeostasis, with excessive bone resorption compared with bone formation, leads to the development of progressive osteopenia leading to lower bone resistance to load, with consequent pain and functional limitations. Phytochemicals with therapeutic and preventive effects against bone resorption have recently received increasing attention since they are potentially more suitable for long-term use than traditional therapeutic chemical compounds. In this systematic review of the literature of the past 5 years, comprehensive information is provided on flavonoids with potential antiresorption and pro-osteogenic effects. It aims to highlight the molecular mechanisms of these mole…

Chromosomal localization and molecular characterization of three different 5S ribosomal DNA clusters in the sea urchin Paracentrotus lividus

In this paper the chromosomal localization and molecular cloning and characterization of three 5S rDNA clusters of 700 bp (base pairs), 900 bp, and 950 bp in the sea urchin Paracentrotus lividus are reported. Southern blot hybridization demonstrated the existence of three 5S rDNA repeats of differing length in the P. lividus genome. Fluorescence in situ hybridization analysis, performed in parallel on both haploid and diploid metaphases and interphase nuclei using different 5S rDNA units as probes, localized these 5S rDNA clusters in 3 different pairs of P. lividus chromosomes. This is the first complete gene mapping not only in a sea urchin but also in the phylum of echinoderms as a whole…

The DNA methylation inhibitor 5-azacytidine modulates 6-thioguanine toxicity in mammalian cells

In order to assess the effects of combining two antimetabolites used separately to treat human leukemias, we carried out an experimental study by exposing V79 Chinese hamster cells, a 6-thioguanine (6-tG)-sensitive cell line, to sequential and concurrent treatments with 5-azacytidine (5-azaC) and 6-tG. In this paper, we demonstrate that there is a clear dependency for the way in which this combination was tested. Pre-treatment with 5-azaC made V79 cells more resistant to 6-tG by a substantial reduction in 6-tG incorporation into DNA; this effect could still be detected for several cell divisions after the removal of 5-azaC, and was achieved neither by reduced cell growth nor by the inductio…

Cytotoxic activity of the histone deacetylase 3-Selective inhibitor Pojamide on MDA-MB-231 triple-negative breast cancer cells

We examined the effects of the ferrocene-based histone deacetylase-3 inhibitor Pojamide (N1-(2-aminophenyl)-N8-ferrocenyloctanediamide) and its two derivatives N1-(2-aminophenyl)-N6-ferrocenyladipamide and N1-(2-aminophenyl)-N8-ferroceniumoctanediamide tetrafluoroborate on triple-negative MDA-MB-231 breast cancer cells. Viability/growth assays indicated that only the first two compounds at 70 &mu

Early and late effects of arsenic exposure in mammalian cells

Previously we demonstrated that V79 Chinese hamster cells underwent either early genetic instability or apoptosis When exposed to sodium arsenite (SA). Genetic instability was evidenced by aneuploid and morphologically abnormal cells, but not by cells with chromosome aberrations. As dividing cells turned out to be the most sensitive to SA exposure, due to the arsenics direct action on the mitotic spindle assembly, we later ascertained the fate of genetically unstable cells escaping apoptosis, by harvesting mitotic rounded-up cells at the end of a 24 h treatment. The progeny of the exposed Chinese hamster cells showed an increased level of mutations related to genome DNA hypomethylation indu…

Genetics and genomics of BNCT among basics and perspectives

The Boron neutron capture therapy (BNCT) bases its therapeutic action on the selective ability to induce DNA damage of tumor cells with the aim to cause their death by apoptosis or necrosis, preserving as much as possible the normal cells. The genetic variability is an evolutionary mechanism of species survival and improvement according to which diversity in genetic configurations exists among individuals of a population, revealed also in different tolerance to insults and reparative ability of DNA damage induced by physical and chemical agents. A tumor, due to its intrinsic genomic instability, acquires the mutator phenotype and may, during the cell duplications, generate in the same indiv…

Dysregulation of DNA methylation induced by past arsenic treatment causes persistent genomic instability in mammalian cells

The mechanisms by which arsenic-induced genomic instability is initiated and maintained are poorly understood. To investigate potential epigenetic mechanisms, in this study we evaluated global DNA methylation levels in V79 cells and human HaCaT keratinocytes at several time points during expanded growth of cell cultures following removal of arsenite exposures. We have found altered genomic methylation patterns that persisted up to 40 cell generations in HaCaT cells after the treatments were withdrawn. Moreover, mRNA expression levels were evaluated by RT-PCR for DNMT1, DNMT3A, DNMT3B, HMLH1, and HMSH2 genes, demonstrating that the down regulation of DNMT3A and DNMT3B genes, but not DNMT1, o…

Bradyrhizobium sp. nodulating the Mediterranean shrub Spanish broom (Spartium junceum L.)

Aims: The molecular diversity of 25 strains of rhizobia, isolated in Sicily from root nodules of the Mediterranean shrubby legume Spanish broom (Spartium junceum L.), is presented in relation to the known rhizobial reference strains. Methods and Results: Our approach to the study of the S. junceum rhizobial diversity combined the information given by the 16S and the intergenic spacer (IGS) 16S–23S rDNA polymorphic region by obtaining them in a single polymerase chain reaction (PCR) step. The PCR fragment size of the S. junceum isolates was 2400–2500 bp and that of the reference strains varied from 2400 in Bradyrhizobium strains to 2800 in Sinorhizobium strains. Inter- and intrageneric lengt…

Early induction of genetic instability and apoptosis by arsenic in cultured Chinese hamster cells

In order to assess at what time from the beginning of exposure inorganic arsenic can give rise to genetic instability and trigger apoptosis, V79-C13 Chinese hamster cells were treated with 10 microM sodium arsenite for 24 h. Under these conditions, cell survival was >70% and cells showed neither an increase in chromosome aberration frequency nor a delay in cell cycle progression. Investigations, which were carried out every 6 h during the treatment, revealed an early appearance of genetically unstable cells, namely micronucleated, multinucleated and mononucleated 'giant' cells, as well as apoptotic cells. Indirect immunostaining using anti-beta-tubulin antibody showed severe alterations in …

Expression levels of PTHrP splicing variants and PTHrP promoter methylation states in differentiating mesenchymal stem cells

An In Vitro Model of Glioma Development

Gliomas are the prevalent forms of brain cancer and derive from glial cells. Among them, astrocytomas are the most frequent. Astrocytes are fundamental for most brain functions, as they contribute to neuronal metabolism and neurotransmission. When they acquire cancer properties, their functions are altered, and, in addition, they start invading the brain parenchyma. Thus, a better knowledge of transformed astrocyte molecular properties is essential. With this aim, we previously developed rat astrocyte clones with increasing cancer properties. In this study, we used proteomic analysis to compare the most transformed clone (A-FC6) with normal primary astrocytes. We found that 154 proteins are…

Il Progetto “Mentori per la didattica” dell’Università di Palermo dopo sei anni dalla nascita e le iniziative future

L’Ateneo di Palermo sta portando avanti diverse iniziative di miglioramento della qualità della didattica. In particolare, il progetto “Mentori per la didattica” nasce nel 2013 su iniziativa di alcuni docenti di ingegneria con l’obiettivo di migliorare la qualità della didattica dei partecipanti grazie a due strumenti chiave: l’aiuto dei mentori e una serie di incontri di riflessione e approfondimento. Ogni docente (mentee) che aderisce al progetto ha due mentori che hanno il compito di aiutarlo a migliorare la qualità della sua didattica. Inoltre, egli stesso svolge il ruolo di mentore a favore di altri due colleghi, cosicché ciascuno è mentore e mentee allo stesso tempo. I mentori assisto…

Effect of gene-gene and gene-environment interactions associated with antituberculosis drug-induced hepatotoxicity.

This study evaluated the association between environmental factors and genetic variations in enzymes that metabolize antituberculosis (anti-TB) drugs [arylamine N-acetyltransferase 2, cytochrome P450 2E1 (CYP2E1), glutathione S-transferase theta 1 (GSTT1), and glutathione S-transferase mu 1] with antituberculosis drug-induced hepatotoxicity (ATDH). We also investigated the potential gene-gene and gene-environment interactions as well as their association with ATDH development in a population of hospitalized TB patients from Buenos Aires.We investigated 364 TB patients who received anti-TB drugs. Physicians collected demographic and clinical data to identify environmental risk factors for AT…

Dysregulation of DNA methylation induced by past arsenic treatment causes persistent genomic instability in mammalian cells

The mechanisms by which arsenic-induced genomic instability is initiated and maintained are poorly understood. To investigate potential epigenetic mechanisms, in this study we evaluated global DNA methylation levels in V79 cells and human HaCaT keratinocytes at several time points during expanded growth of cell cultures following removal of arsenite exposures. We have found altered genomic methylation patterns that persisted up to 40 cell generations in HaCaT cells after the treatments were withdrawn. Moreover, mRNA expression levels were evaluated by RT-PCR for DNMT1, DNMT3A, DNMT3B, HMLH1, and HMSH2 genes, demonstrating that the down regulation of DNMT3A and DNMT3B genes, but not DNMT1, o…

Genotoxicity and Epigenotoxicity of Carbazole-Derived Molecules on MCF-7 Breast Cancer Cells

The carbazole compounds PK9320 (1-(9-ethyl-7-(furan-2-yl)-9H-carbazol-3-yl)-N-methylmethanamine) and PK9323 (1-(9-ethyl-7-(thiazol-4-yl)-9H-carbazol-3-yl)-N-methylmethanamine), second-generation analogues of PK083 (1-(9-ethyl-9H-carbazol-3-yl)-N-methylmethanamine), restore p53 signaling in Y220C p53-mutated cancer cells by binding to a mutation-induced surface crevice and acting as molecular chaperones. In the present paper, these three molecules have been tested for mutant p53-independent genotoxic and epigenomic effects on wild-type p53 MCF-7 breast adenocarcinoma cells, employing a combination of Western blot for phospho-γH2AX histone, Comet assay and methylation-sensitive arbitrarily pr…

Global diversity in the TAS2R38 bitter taste receptor: revisiting a classic evolutionary PROPosal

AbstractThe ability to taste phenylthiocarbamide (PTC) and 6-n-propylthiouracil (PROP) is a polymorphic trait mediated by the TAS2R38 bitter taste receptor gene. It has long been hypothesized that global genetic diversity at this locus evolved under pervasive pressures from balancing natural selection. However, recent high-resolution population genetic studies of TAS2Rs suggest that demographic events have played a critical role in the evolution of these genes. We here utilized the largest TAS2R38 database yet analyzed, consisting of 5,589 individuals from 105 populations, to examine natural selection, haplotype frequencies and linkage disequilibrium to estimate the effects of both selectio…

Due diverse esperienze di lezioni sperimentali condotte secondo il metodo di co-teaching

Nell’ambito del progetto “Mentori per la didattica”, nato nel 2013 da un ristretto gruppo di docenti dell’Università di Palermo e che comprende oggi 120 docenti, oltre alle attività di mentoring fra pari, sono state promosse diverse attività formative. Fra queste, tre workshop in tre anni consecutivi sono stati dedicati alla formazione sull’active learning e su metodi e tecniche di didattica innovativa ed in particolare nell’ultimo anno è stata importante la partecipazione di alcuni componenti del progetto al congresso “Faculty development“ e al pre-workshop del convegno di Genova di maggio 2019. A partire da tutte queste esperienze, diversi docenti hanno iniziato a sperimentare nuove modal…

Genotoxicity of Terpenes Present in Wastewater of a Citrus Transformation Factory in Bacterial and Mammalian Cells and Effectiveness of Photocatalytic Degradation

The aim of this work was to compare the genotoxic responses of mixtures of terpenes present in wastewaters of a citrus transformation factory with the genotoxicity of the individual compounds. Samplings of wastewater collected before (untreated sample) and past water purification by biological method (treated sample) were analyzed using Solid Phase Micro-extraction (SPME) followed by GC analyses. The chromatograms showed in all effluents the presence of four terpenes: pinene, -pinene, 3-carene, D-limonene. The concentrations of terpenes in the untreated sample were 1–3 orders of magnitude higher than in the treated sample. Genotoxicity was evaluated in the Salmonella reversion assay (Ames t…

Anti-proliferative and pro-apoptotic activity of whole extract and isolated indicaxanthin from Opuntia ficus-indica associated with re-activation of the onco-suppressor p16(INK4a) gene in human colorectal carcinoma (Caco-2) cells.

Phytochemicals may exert chemo-preventive effects on cells of the gastro-intestinal tract by modulating epigenome-regulated gene expression. The effect of the aqueous extract from the edible fruit of Opuntia ficus-indica (OFI extract), and of its betalain pigment indicaxanthin (Ind), on proliferation of human colon cancer Caco-2 cells has been investigated. Whole extract and Ind caused a dose-dependent apoptosis of proliferating cells at nutritionally relevant amounts, with IC50 400 ± 25 mg fresh pulp equivalents/mL, and 115 ± 15 μM (n = 9), respectively, without toxicity for post-confluent differentiated cells. Ind accounted for ∼80% of the effect of the whole extract. Ind did not cause ox…

Mid-region parathyroid hormone-related protein is a genome-wide chromatin-binding factor that promotes growth and differentiation of HB2 epithelial cells from the human breast

Human parathyroid hormone-related protein (PTHrP) is a polyhormone that undergoes proteolytic cleavage producing smaller peptides which exert diversified biological effects. PTHrP signalization is prominently involved in breast development and physiology, but the studies have been focused onto either N-terminal species or full-length protein introduced by gene transfer techniques. Our present work investigates for the first time the effect of the mid-region PTHrP secretory form, that is, the fragment [38-94], on HB2 non-tumoral breast epithelial cells. We examined viability/proliferation of cells grown in PTHrP-containing media supplemented with different serum concentration and on differen…

Karyotype Abnormalities in a Variant Chinese Hamster Cell Line Resistant to Methyl Methanesulphonate

A variant cell population, isolated from V79-CI 3 Chinese hamster cells after two consecutive treatments with methyl methanesulphonate (MMS), was found to be highly resistant to killing by this alkylating agent. The resistant cell line was cytogenetically characterized both by the presence of a stable translocation involving metacentric chromosome 2 and acrocentric chromosome 6 and by a supernumerary chromosome originated by the duplication of a small telocentric chromosome. This cell population also showed a transient transformed phenotype, seen as formation of transformed foci containing cells with high chromosomes counts and multiple chromosomal aberrations. As MMS-resistance and karyoty…

Molecular Signatures Associated with Treatment of Triple-Negative MDA-MB231 Breast Cancer Cells with Histone Deacetylase Inhibitors JAHA and SAHA

Jay Amin Hydroxamic Acid (JAHA; N8-ferrocenylN1-hydroxy-octanediamide) is a ferrocene-containing analogue of the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA). JAHA’s cytotoxic activity on MDA-MB231 triple negative breast cancer (TNBC) cells at 72 h has been previously demonstrated with an IC50 of 8.45 M. JAHA’s lethal effect was found linked to perturbations of cell cycle, mitochondrial activity, signal transduction and autophagy mechanisms. In order to glean novel insights on how MDA-MB231 breast cancer cells respond to the cytotoxic effect induced by JAHA, and to compare the biological effect with the related compound SAHA, we have employed a combination of…

Variability in bitter taste perception and correlation with SNP in TAS2R38 gene in different student population

People vary widely in their sensitivities to bitter compounds. The PTC gene, TAS2R38 on chromosome 7, consists a single coding exon which encodes a G-protein-coupled receptor. The taster subject contains a proline at position 49, an alanine at position 262. and a valine at position 296 (PAV form), while the no taster contains an alanine, a valine, and an isoleucine at these 3 positions, respectively (AVI form).These two forms depend on three single nucleotide polymorphisms (SNP). By providing students with paper soaked in different solutions of PTC it was been possible to assess the individual threshold of bitterness sensitivity. DNA was extracted from saliva, and following PCR amplification…

Protective effects of melatonin in inflamed intestinal epithelium are associated with reduced NF-κB activation and changes in DNA methylation status

Melatonin is the main product of the pineal gland but is also released in the gastrointestinal tract (GIT). Production of melatonin at GIT is independent of the photoperiod and contributes almost completely to plasma melatonin concentration during daylight hours. The physiological role of melatonin at GIT is poorly characterized but recently anti-inflammatory effects have been reported. In this study, we evaluated the effect of Melatonin in intestinal epithelial cells (IEC) stimulated by Interleukin-1β. Our results clearly show that melatonin at micromolar concentrations inhibits the inflammatory response in IEC. The protective effect is expressed through a marked decrease in release and ex…

Terpenoid treatment in osteoporosis: this is where we have come in research.

Lower bone resistance to load is due to the imbalance of bone homeostasis, where excessive bone resorption, compared with bone formation, determines a progressive osteopenia, leading to a high risk of fractures and consequent pain and functional limitations. Terpenoids, with their activities against bone resorption, have recently received increased attention from researchers. They are potentially more suitable for long-term use compared with traditional therapeutics. In this review of the literature of the past 5 years, we provide comprehensive information on terpenoids, with their anti-osteoporotic effects, highlighting molecular mechanisms that are often in epigenetic key and a possible p…

Functional variants of 5S rRNA in the ribosomes of common sea urchin Paracentrotus lividus

Abstract We have previously reported a molecular and cytogenetic characterization of three different 5S rDNA clusters in the sea urchin Paracentrotus lividus ; this study, performed at DNA level only, lends itself as starting point to verify that these clusters could contain transcribed genes, then, to demonstrate the presence of heterogeneity at functional RNA level, also. In the present work we report in P. lividus ribosomes the existence of several transcribed variants of the 5S rRNA and we associate all transcribed variants to the cluster to which belong. Our finding is the first demonstration of the presence of high heterogeneity in functional 5S rRNA molecules in animal ribosomes, a f…

Scienze Naturali

Il corso è stato progettato per disporre di tutti gli argomenti previsti da programma ministeriale per il quinto anno dei Licei (Chimica organica, Genetica, Biochimica, Microbiologia, Biotecnologie e Scienze della Terra) in un unico volume. Gli argomenti proposti sono trattati in maniera organica, evidenziando i possibili punti di contatto tra le differenti discipline e abituando lo studente a sviluppare una concezione liquida del sapere. La didattica e la struttura proposte sono finalizzate alla conoscenza e alla comprensione dei fenomeni che contraddistinguono la materia, la natura, gli esseri viventi e l’uomo e l’ambiente nel quale vive e col quale interagisce. Il corso concorre alla cos…

CHARACTERIZATION OF TRANSFORMED CELL LINES OBTAINED FROM PRIMARY RAT CORTICAL ASTROCYTES

Brain cancers are complex and heterogeneous; most of them derive from glial cells[1], and are called gliomas, further subdivided into astrocytomas, oligodendrogliomas, ependymomas and glioastrocytomas[2]. The malignant cells undergo modifications of their metabolism and behaviour, and acquire the ability to migrate along the blood vessels in small groups (model of the guerrilla war)[3], thus invading the surrounding brain parenchyma. Most important, they have the capacity to affect the surrounding microenvironment, by altering both the extracellular matrix and the properties of the normal cells present in the brain, including glial-, endothelial-, and immune-cells, further promoting cancer …

Dalla Chimica alla Biochimica

Come molecole semplici possono formare molecole complesse e come queste ultime possono addirittura dare origine ad esseri viventi come un batterio o addirittura un essere umano? Cosa rende i viventi profondamente diversi da un computer o da una semplice roccia? In questo capitolo assisterete ad una affascinante svolta nella storia evolutiva del nostro pianeta accaduta molti milioni di anni fa: la chimica del mondo non ancora vivente si evolve per tappe e fonda l’unità elementare chiamata cellula: la prima aggregazione di molecole chimiche che può essere definita “vivente”. Scoprirete come grazie alla termodinamica, che vige in una cellula come altrove, può avvenire un insieme vastissimo di …

Le razze umane non esistono, lo dimostra…. il colore della pelle!

Alcune manifestazioni fenotipiche della specie umana sono tramandate di generazione in generazione secondo delle regole che apparentemente non seguono le classiche leggi di Mendel: sono i caratteri continui, un tipo particolare di ereditarietà in cui ha molta influenza l’ambiente ed anche le sue interazioni con il genoma. Solo per questi tipi particolari di caratteri è possibile definire due grandezze, l’ereditabilità e la varianza, che danno una misura di quanta sia la componente genetica e di quanto quella ambientale nella loro definizione. Studi di correlazione fra persone non imparentate e gemelli monozigoti hanno dimostrato che il carattere continuo umano “creste digitali” ha un alto v…

IL-1. induces DNA demethylation at genome level and in specific CpG sites of IL-6 and IL-8 genes in human intestinal epithelial cells

Inflammation is a complex physiological response that requires the activity of a sophisticated regulatory network involving the activation of specific genes for defense, tissue repair and remodeling. Although transcriptional activation has been shown to be critical in the regulation of inflammatory genes (1) the role of epigenetic phenomena in the modulation of the inflammatory response is now emerging (2). Specifically, it has been recently reported that proinflammatory stimuli induce DNA demethylation in the interleukin IL-1 promoter of human articular chondrocytes (3). IL-1 cytokine, among several proinflammatory agents, represents an essential player in the inflammatory conditions of …

Mid-region parathyroid hormone-related protein (PTHrP) binds chromatin of MDA-MB231 breast cancer cells and isolated oligonucleotides “in vitro”

We have previously shown that PTHrP(38-94)-amide restrains growth and invasion "in vitro", causes striking toxicity and accelerates death of some breast cancer cell lines, the most responsive being MDA-MB231 whose tumorigenesis was also attenuated "in vivo". PTHrP(38-94)-amide contains the domain implicated in the nuclear import of PTHrP. Although the nucleus was identified as a destination for mid-region PTHrP, evidence for direct DNA-binding capability is lacking to date. Here, we examined the localization of PTHrP(38-94)-amide within MDA-MB231 cells and within metaphase spread preparations and characterized its DNA-binding properties, employing a combination of immunocytochemical, cytoge…

Chemical Profile and Biological Activity of Cherimoya (Annona cherimola Mill.) and Atemoya (Annona atemoya) Leaves

Annona cherimola (Cherimoya) and Annona atemoya (Atemoya) are tropical plants known for their edible fruit. Scientific data suggest that their leaves, used in traditional medicine in the form of teas or infusions without evidence of toxicity, contain several bioactive compounds. However, only Annona muricata among all the Annona species is currently used in the nutraceutical field, and its dried leaves are marketed for tea preparation. In this work, we explored the nutraceutical potential of Atemoya and Cherimoya leaves, by evaluating their chemical profile and functional properties. Phytochemical analyses showed large amounts of phenolic compounds, in particular proanthocyanidins, and iden…

Nutritional epigenomic and DNA-damage modulation effect of natural stilbenoids

The aim of the present work is the evaluation of biological effects of natural stilbenoids found in Vitis vinifera, with a focus on their activity as epigenetic modulators. In the present study, resveratrol, pterostilbene and for the first time their dimers (±)-trans-δ-viniferin, (±)-trans-pterostilbene dehydrodimer were evaluated in Caco-2 and HepG-2 cell lines as potential epigenetic modulators. Stilbenoids were added in a Caco-2 cell culture as a model of the intestinal epithelial barrier and in the HepG-2 as a model of hepatic environment, to verify their dose-dependent toxicity, ability to interact with DNA, and epigenomic action. Resveratrol, pterostilbene, and (±)-trans-pterostilbene…

Water: The First Archaic Mutagen of Evolution, the Adoptive Mother of Currently Nucleic Acids

It would be intuitive to think that for the evolutionary onset of nucleic acids, and even before of their monomeric constituents, the water was crucial. Within the series of evolutions which start from the origin of the universe and still cannot, by definition, be considered concluded, certainly among the geothermal evolution end and the beginning of the chemical one, on Earth, the moment was favorable for the arrival of the first proto-nucleotides: from underground deposits of methanehydrate [1] and phosphate, with the support of all known pre-biotic physical-chemical conditions, were made the monomeric components of nucleic acids. The cradle of nucleic acids does not seem to have been so …

Telomere dysfunction in cells with arsenic-induced genomic instability

It is well known that the occurrence of dicentric chromosomes represent signature of telomere dysfunction and is a clear symptom of genomic instability. V79 Chinese hamster cells, treated with 10µM sodium arsenite for 24h and allowed to grow in drug-free medium (ASO cells), showed genomic instability with aneuploidy and nuclear abnormalities as well as the appearance of dicentric chromosomes since the 90th cell generation. TRAP assay was performed on growing ASO cells and on clones isolated during the course of the expanded growth. As expected, some clones with dicentric chromosomes and severely reduced telomerase activity went to death; surprisingly, other clones also bearing chromosomal e…

The role of Plant Growth Promoting (PGP) bacteria in the growth of Mediterranean diet plants producing high-value compounds

Loss of fertility, together with drought and aridity, results in soil unproductivity and eventually desertification. Unsustainable agricultural practices are currently stressing the soil: an undervalued resource that, in addition to food, provides a multitude of invaluable services such as regulating the microclimate, reducing contaminants, and capturing CO2. Hence, there is an increasing need to resort to sustainable approaches toward agricultural systems. Although Sicily is one of those regions most affected by water shortages, it is at the same time a reservoir of drought-resistant Mediterranean plants with high nutritional value and a rich microbiota. This project aims to promote agricu…

Radiazione solare e variabilità genetica: cosa ci regala il sole sulla spiaggia (oltre la tintarella)

La radiazione solare ha una componente UV ed è ormai abbastanza noto che questi raggi, essendo radiazioni ionizzanti, procurano danni al DNA. Più in particolare, l’energia conferita da queste radiazioni a due o più basi azotate pirimidiniche adiacenti su una stessa emielica di DNA, forma i “dimeri di pirimidina”, tipico addotto che renderebbe il DNA localmente illeggibile e non replicabile se non eliminato dai sistemi endogeni di riparazione. Ma per fortuna, in una cellula umana ci sono molti complessi enzimatici deputati alla rimozione dei dimeri di pirimidina e, considerando che ogni enzima è sempre codificato da un gene e che per ogni gene, per variabilità genetica, possono esistere tant…

Role of the antioxidant defence system and telomerase in arsenic-induced genomic instability

Arsenic (AS) is a reactive oxygen species (ROS)-inducer carcinogen, whose mode of action is still unclear. To defend against ROS, cells use enzymatic and non-enzymatic antioxidants, such as superoxide dismutase (SOD) and catalase. Failure of antioxidant systems (AXS) can result in dicentric chromosomes formation as well as telomere associations for the reduced activity of telomerase. In order to clarify the long-term effects of a past AS exposure, we evaluated the efficiency of the AXS and the telomerase activity in the progeny of arsenite-treated cells named ASO (arsenic shake-off) cells, previously obtained from arsenite-treated V79 cells and selected by shake-off. Despite SOD1 expression…

Long-Lasting Genomic Instability Following Arsenite Exposure inMammalian Cells: The Role of Reactive Oxygen Species

Previously, we reported that the progeny of mammalian cells, which has been exposed to sodium arsenite for two cell cycles, exhibited chromosomal instability and concurrent DNA hypomethylation, when they were subsequently investigated after two months of subculturing (about 120 cell generations) in arsenite-free medium. In this work, we continued our investigations of the long-lasting arsenite-induced genomic instability by analyzing additional endpoints at several time points during the cell expanded growth. In addition to the progressive increase of aneuploid cells, we also noted micronucleated and multinucleated cells that continued to accumulate up to the 50th cell generation, as well a…

Fifteen years’ experience treating cells with inorganic arsenic, a molecule able to induce genetic / genomic instability and epigenetic changes even after its removal

Treating V79-Cl3 cells with 10 µM sodium arsenite (SA) for 24h we observed severe alterations in spindle morphology and aneuploidy; treating rat astrocytes with SA we detected HSP70 induction and DNA damage. We assumed that SA induced in dividing cells early genetic instability. Subsequently, we in vitro stabilized those V79-Cl3 cells dividing at the end of SA-treatment and maintained for long without SA (ASO cells). In ASO cells, we observed chromosomal rearrangements, increased spontaneous mutations, genome-wide DNA hypomethylation (GWDH), similarly to exposed cells. We inferred that a short-term SA exposure has long-term effects and that GWDH enhances the genetic instability. Consequentl…

Intergenerational Effect of Early Life Exposure to Permethrin: Changes in Global DNA Methylation and in Nurr1 Gene Expression

Environmental exposure to pesticides during the early stages of development represents an important risk factor for the onset of neurodegenerative diseases in adult age. Neonatal exposure to Permethrin (PERM), a member of the family of synthetic pyrethroids, can induce a Parkinson-like disease and cause some alterations in striatum of rats, involving both genetic and epigenetic pathways. Through gene expression analysis and global DNA methylation assessment in both PERM-treated parents and their untreated offspring, we investigated on the prospective intergenerational effect of this pesticide. Thirty-three percent of progeny presents the same Nurr1 alteration as rats exposed to permethrin i…

Panta rei: la genetica si racconta

CYP2E1 VNTR polymorphisms and hepatocarcinoma: a gender-specific correlation

Cytochrome P450 (CYP2E1) is often associate to susceptibility to alcohol-related diseases and various cancers, because of its role in the metabolism of multiple environmental xenobiotics. In the 5’- flanking region of the human CYP2E1 gene there are restriction fragment length polymorphism which are involved in the transcriptional regulation of the CYP2E1 gene. Recently a tandem repeat polymorphism (VNTR) in the 5’-flanking region of CYP2E1 was found. Because cytochrome P450 2E1 catalyzes the metabolic activation of pro-carcinogen and cytotoxic compound, we value the genetic distribution of this tandem repeat polymorphism in a healthy population, and in patients with hepatocellular carcinom…

Establishment and Preliminary Characterization of Three Astrocytic Cells Lines Obtained from Primary Rat Astrocytes by Sub-Cloning.

Gliomas are complex and heterogeneous tumors that originate from the glial cells of the brain. The malignant cells undergo deep modifications of their metabolism, and acquire the capacity to invade the brain parenchyma and to induce epigenetic modifications in the other brain cell types. In spite of the efforts made to define the pathology at the molecular level, and to set novel approaches to reach the infiltrating cells, gliomas are still fatal. In order to gain a better knowledge of the cellular events that accompany astrocyte transformation, we developed three increasingly transformed astrocyte cell lines, starting from primary rat cortical astrocytes, and analyzed them at the cytogenet…

Sister chromatid exchange in Waldenström's macroglobulinemia

Results on sister chromatid exchange (SCE) frequency and interchromosomal distribution in bone marrow and peripheral blood cultures from patients with Waldenström's macroglobulinemia are reported. PHA-stimulated bone marrow cultures showed increased SCE frequencies in all 12 patients examined. The increase was particularly high in two cases (17.07 and 16.77 SCE/cell, respectively) and, in one of them, a very high SCE level was found in PHA-stimulated peripheral blood culture (40.81 SCE/cell). In LPS-stimulated cultures, increased SCE levels were observed in some patients. Comparison between SCE frequency in bone marrow cell cultures with either mitogen showed a significant increase in PHA-s…

PTHrP isoform expression in adipo- and osteo-differentiating human mesenchymal stem cells

Variable Number of Tandem Repeats (VNTR) gene polymorphism of CYP2E1 in patients with pancreatic adenocarcinoma

Context: The genetic polymorphism is considered a major source of variability, influencing the levels of gene expression. Cytochrome P450 2E1 (CYP2E1) is a mixed-function oxidase involved in the metabolism of the many endogenous and exogenous substances (ethanol, chemical carcinogens) in the hepatic and pancreatic tissue. CYP2E1 gene polymorphisms can cause various abilities of metabolize xenobiotic substances within a population with consequent increased susceptibility to various diseases,including cancer. One of the polymorphisms of the CYP2E1 gene is a VNTR (Variable Number Tandem Repeat) of some sequences in its "5 '- flanking region. Method : VNTR genotype CYP2E1 was determined by RFLP…

Effect of inorganic arsenic on rat cortical astrocytes in culture

Although inorganic arsenic is a well known poisonous metalloid, the cellular and molecular mechanisms of its action remain elusive. The present study was aimed at analyzing the effects of NaAsO2 on primary cultures of rat astrocytes by determining DNA damage by comet assay, and by evaluating possible changes of the concentration of some conserved heat shock proteins. Cells treated with inorganic arsenic underwent induction of Hsp70, demonstrating a state of stress. Moreover, although micromolar NaAsO2 treatments (60 μM) only reduced cell viability to 60% respect to untreated cells, high DNA damage was already observed after 24h treatment with 10 μM arsenite. Since arsenic is known to be not…

Melatonin reduces inflammatory response in human intestinal epithelial cells stimulated by interleukin‐1β

Melatonin is the main secretory product of the pineal gland, and it is involved in the regulation of periodic events. A melatonin production independent of the photoperiod is typical of the gut. However, the local physiological role of melatonin at the intestinal tract is poorly characterized. In this study, we evaluated the anti-inflammatory activities of melatonin in an in vitro model of inflamed intestinal epithelium. To this purpose, we assessed different parameters usually associated with intestinal inflammation using IL-1 beta-stimulated Caco-2 cells. Differentiated monolayers of Caco-2 cells were preincubated with melatonin (1 nmol/L-50 mu mol/L) and then exposed to IL-1 beta. After …

Antiproliferative and pro-apoptotic effects of the phytochemical Indicaxanthin on human intestinal (Caco-2) and hepatic (Ha 22T) cancer cell lines

In the present study antiproliferative effects of Indicaxanthin (Ind), a highly bioavailable pigment from the fruits of Opuntia ficus-indica (1), were investigated on a number of human cancer cell lines including hepatocarcinoma cells (HepG2, Ha22T, HUH 7), breast cancer cells (MCF7), cervix epithelial carcinoma (HeLa), and colorectal carcinoma cells (Caco-2). Cytotoxicity of Ind, in a concentration range between 25 to 100 M, was evaluated by Trypan blue exclusion method and MTT assay. Ind caused a clear dose- and time-dependent decrease in the proliferation of Caco-2 and Ha 22T cells with an IC(50) of about 50 M, with minor effect on the other cell lines. Flow cytometric analysis after A…

In vivo and in vitro inhibitory effects of acrylamide on DNA topoisomerase II

Acrylamide (AA), a chemical produced in several foodstuffs when cooked at a high temperature, is considered a probable human carcinogen, but the molecular mechanism underlying its genotoxicity has not fully known. Numerous authors have reported the induction by AA of DNA double strand breaks and sister chromatid exchange (SCE); we here confirmed the acrylamide capability of damaging DNA by utilizing Comet assay, which showed a dose-dependent increase of tail lenght, in metabolically non competent V79 Chinese hamster cells. Moreover, we observed that Acrylamide (AA) was able to antagonize in vivo the citotoxicity of well know poison etoposide; this suggested that topoisomerase II activity wa…