Centrosome amplification induced by hydroxyurea leads to aneuploidy in pRB deficient human and mouse fibroblasts.

Alterations in the number and/or morphology of centrosomes are frequently observed in human tumours. However, it is still debated if a direct link between supernumerary centrosomes and tumorigenesis exists and if centrosome amplification could directly cause aneuploidy. Here, we report that hydroxyurea treatment induced centrosome amplification in both human fibroblasts expressing the HPV16 -E6-E7 oncoproteins, which act principally by targeting p53 and pRB, respectively, and in conditional pRB deficient mouse fibroblasts. Following hydroxyurea removal both normal and p53 deficient human fibroblasts arrested. On the contrary pRB deficient fibroblasts entered the cell cycle generating aneupl…

DNA Methyltransferase1 post-transcriptional silencing induces aneuploidy and cell cycle arrest in human cells.

The regulation of chromatin structure is a dynamic and complex process that is modulated by epigenetic mechanisms. Malfunctioning of these processes can cause gene expression alteration and could compromise important events such as chromosome condensation and segregation. Imbalance in cytosine methylation and deregulation of DNA-methyltransferases (DNMTs), and of DNMT1 in particular, is frequent in human cancers. To investigate DNMT1 implication in the generation of aneuploidy we evaluated the effects of its depletion by RNA-interference both in primary human cells (IMR90) and in near diploid human tumor (HCT116) cells. Posttranscriptional silencing of DNMT1 induced aneuploidy, cell prolife…

MAD2 depletion triggers premature cellular senescence in human primary fibroblasts by activating a P53 pathway preventing aneuploid cells propagation.

The spindle assembly checkpoint (SAC) is a cellular surveillance mechanism that ensures faithful chromosome segregation during mitosis and its failure can result in aneuploidy. Previously, it was suggested that reduction of the MAD2 gene, encoding a major component of the SAC, induced aneuploidy in human tumor cells. However, tumor cell lines contain multiple mutations that might affect or exacerbate the cellular response to Mad2 depletion. Thus, the scenario resulting by Mad2 depletion in primary human cells could be different and more complex that the one depicted so far. We used primary human fibroblasts (IMR90) and epithelial breast cells (MCF10A) to gain further insight on the effects …

Premature termination codon 124 derivatives as a novel approach to improve the read-through of premature amber and ochre stop codons

Nucleotide changes within an exon may alter the trinucleotide normally encoding a particular amino acid, such that a new stop signal is transcribed into the mRNA open reading frame. A recent approach to directly overcome the deleterious effects caused by nonsense mutations is represented by readthrough strategies which take advantage of the known properties of aminoglycosides that can suppress stop codons.

Nonsense codons suppression. An acute toxicity study of three optimized TRIDs in murine model, safety and tolerability evaluation.

Stop mutations cause 11% of the genetic diseases, due to the introduction of a premature termination codon (PTC) in the mRNA, followed by the production of a truncated protein. A promising therapeutic approach is the suppression therapy by Translational Readthrough Inducing Drugs (TRIDs), restoring the expression of the protein. Recently, three new TRIDs (NV848, NV914, NV930) have been proposed, and validated by several in vitro assays, for the rescue of the CFTR protein, involved in Cystic Fibrosis disease. In this work, an acute toxicological study for the three TRIDs was conducted in vivo on mice, according to the OECD No.420 guidelines. Animals were divided into groups and treated with …

COMBINING TRANSLATION READTHROUGH INDUCING DRUGS AND NONSENSE MEDIATED DECAY PATWHAY INHIBITION TO THE CFTR RESCUE IN CYSTIC FIBROSIS CELL MODEL SYSTEM

Nonsense mutations affect 10% of patients with cystic fibrosis and produce a premature termination codon in CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) mRNA causing early termination of translation and leading to lack of CFTR function. A potential therapy for nonsense mutations provides the use of small molecules able to overcome the premature stop codon (PTC) by a readthrough mechanism that lead to synthesis a complete CFTR protein. Despite the good results obtained from this approach, TRIDs efficiency is considerably reduced by the poor amount of target transcript, that is the mRNA containing the PTC. The readthrough, indeed, does not occur on the totality of target transcr…

RESCUE OF LRBA GENE EXPRESSION IN PRIMARY HUMAN FIBROBLASTS CHARACTERISED BY NONSENSE MUTATION c. 5047 (C>T).

Primary immunodeficiencies (PIDs) are rare genetic diseases characterized by susceptibility to infections, increased risk of autoimmunity, hypogammaglobulinemia, and lymphoproliferative syndromes. PIDs are associated to genetic alterations in about 400 known genes, among which, mutations of the LRBA gene. LRBA gene encodes a widely expressed multi-domain protein with highly conserved BEACH domain, involved in regulation of endosomal trafficking, particularly endocytosis of ligand-activated receptors. It was reported that stop mutations affect this gene leading to the loss of the protein expression. Recently, we identified three Translational Readthrough Inducing Drug (TRID), that showed hig…

Chromosomal instability promoted by RB depletion relied neither on p53 nor SAC dysfunction in HCT116 tumor cells

p14ARF re-expression induces apoptosis in aneuploid HCT116 cells

Weakening the Spindle Assembly Checkpoint by reduced expression of its components such as MAD2, BubR1 and MPS1 induces chromosome instability and aneuploidy both hallmarks of cancer cells. p14ARF that is found frequently altered in human cancers, is overexpressed in response to oncogenic stimuli to stabilize p53 halting cell progression. Previously, we determined that lack or reduced expression of p14ARF is involved in the maintenance of aneuploid cells suggesting that it could be part of a pathway controlling proliferation of aneuploidy cells. To investigate further this aspect of p14ARF function it was ectopically expressed in HCT116 cells, a stable near diploid cell line, after MAD2 depl…

Hydroxyurea induces centrosome amplification and slowing down of cell cycle in colo-cancer cells SW480

Bypass of G1 arrest induced by DNMT1 posttranscriptional silencing triggers aneuploidy in human cells.

Aneuploidy is a major source of genomic instability in cancer, resulting from chromosome segregation errors caused by defects in genes controlling correct mitotic spindle assembly, centrosome duplication and cell cycle checkpoints. Interestingly in aneuploid cells some of these genes, although not mutated, were underexpressed suggesting the involvement of epigenetic alterations. DNA methylation and histone modifications are the main epigenetic modifications occurring in cells. DNA methyl-transferase 1 (Dnmt1) is known to restore DNA methylation patterns during cell divisions. We investigated the effects of DNMT1 silencing by RNA-interference on the generation of aneuploidy in primary human …

Identification and validation of novel molecules obtained by integrated computational and experimental approaches for the read-through of PTCs in CF cells

Effetti del Silenziamento dei geni Plk1, Brca1 e Rb nella duplicazione dei centrosomi in cellule tumorali HCT116

Acute loss of retinoblastoma function induces centrosomes amplification both in murine and human fibroblasts

Does GLP-2 receptor expression change dynamically in fed and fasted states?

Deciphering the Nonsense Readthrough Mechanism of Action of Ataluren: An in Silico Compared Study

Ataluren was reported to suppress nonsense mutations by promoting the readthrough of premature stop codons, although its mechanism of action (MOA) is still debated. The likely interaction of Ataluren with CFTR-mRNA has been previously studied by molecular dynamics. In this work we extended the modeling of Ataluren's MOA by complementary computational approaches such as induced fit docking (IFD), quantum polarized ligand docking (QPLD), MM-GBSA free-energy calculations, and computational mutagenesis. In addition to CFTR-mRNA, this study considered other model targets implicated in the translation process, such as eukaryotic rRNA 18S, prokaryotic rRNA 16S, and eukaryotic Release Factor 1 (eRF…

RB acute loss affects expression of mitotic genes and is associated with centrosome amplification and aneuploidy

Gastric relaxation induced by glucagon-like peptide-2 and receptorial expression in fasted or high-fat diet fed mice.

Retinoblastoma epidemiology: Does the evidence matter?

It has been proposed that retinoblastoma is 'caused' by two sequential mutations affecting the RB1 gene, but this is a rather outdated view of cancer aetiology that does not take into account a large amount of new acquisitions such as chromosomal and epigenetic alterations. Retinoblastoma remains probably the only cancer in which the rather simplistic 'two hit' mutational model is still considered of value, although cancer is known to be associated with genomic and microsatellite instability, defects of the DNA mismatch repair system, alterations of DNA methylation and hystone acethylation/deacethylation, and aneuploidy. Moreover, as it is shown herein, the predictions made by the 'two hit'…

Caffeine boosts Ataluren's readthrough activity

Abstract The readthrough of nonsense mutations by small molecules like Ataluren is considered a novel therapeutic approach to overcome the gene defect in several genetic diseases as cystic fibrosis (CF). This pharmacological approach suppresses translation termination at premature termination codons (PTCs readthrough) thus restoring the expression of a functional protein. However, readthrough might be limited by the nonsense-mediated mRNA decay (NMD), a cell process that reduces the amount/level of PTCs containing mRNAs. Here we investigate the combined action of Ataluren and caffeine to enhance the readthrough of PTCs. IB3.1 CF cells with a nonsense mutation were treated with caffeine to a…

Identification of a new molecule with readthrough activity to rescue CFTR protein function

In Cystic fibrosis (CF) disease nonsense mutations in the CFTR gene cause absence of the CFTR protein expression and a more severe form of the disease. About 10% of patient affected by CF show a nonsense mutation. A potential treatment of this alteration is to promote translational readthrough of premature termination codons (PTCs) by translational readthrough inducing drugs such as Ataluren. In this context we aimed to compare the 1,2,4-oxadiazole core of Ataluren with a slightly different scaffold, the 1,3,4oxadiazole core. By a validated protocol consisting of computational screening, synthesis and biological tests we identified, a new small molecule with 1,3,4-oxadiazole core (2a/NV2445…

RNAi mediated acute depletion of Retinoblastoma protein (pRb) promotes aneuploidy in human primary cells via micronuclei formation

BACKGROUND: Changes in chromosome number or structure as well as supernumerary centrosomes and multipolar mitoses are commonly observed in human tumors. Thus, centrosome amplification and mitotic checkpoint dysfunctions are believed possible causes of chromosomal instability. The Retinoblastoma tumor suppressor (RB) participates in the regulation of synchrony between DNA synthesis and centrosome duplication and it is involved in transcription regulation of some mitotic genes. Primary human fibroblasts were transfected transiently with short interfering RNA (siRNA) specific for human pRb to investigate the effects of pRb acute loss on chromosomal stability. RESULTS: Acutely pRb-depleted fibr…

Enhancement of premature stop codon readthrough in the CFTR gene by Ataluren (PTC124) derivatives.

Abstract Premature stop codons are the result of nonsense mutations occurring within the coding sequence of a gene. These mutations lead to the synthesis of a truncated protein and are responsible for several genetic diseases. A potential pharmacological approach to treat these diseases is to promote the translational readthrough of premature stop codons by small molecules aiming to restore the full-length protein. The compound PTC124 (Ataluren) was reported to promote the readthrough of the premature UGA stop codon, although its activity was questioned. The potential interaction of PTC124 with mutated mRNA was recently suggested by molecular dynamics (MD) studies highlighting the importanc…

Acute loss of retinoblastoma function induces centrosome amplification and aneuploidy both in human and murine primary fibroblasts

Simultaneous Aurora-A/STK15 overexpression and centrosome amplification induce chromosomal instability in tumour cells with a MIN phenotype

Abstract Background Genetic instability is a hallmark of tumours and preneoplastic lesions. The predominant form of genome instability in human cancer is chromosome instability (CIN). CIN is characterized by chromosomal aberrations, gains or losses of whole chromosomes (aneuploidy), and it is often associated with centrosome amplification. Centrosomes control cell division by forming a bipolar mitotic spindle and play an essential role in the maintenance of chromosomal stability. However, whether centrosome amplification could directly cause aneuploidy is not fully established. Also, alterations in genes required for mitotic progression could be involved in CIN. A major candidate is represe…

PTC124 derivatives as a novel approach to improve the readthrough of premature stop codons in the CFTR gene.

Background Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). Approximately 10% (worldwide) of patients have in-frame nonsense mutations (UAA, UAG or UGA class I mutations) in the CF trans-membrane regulator (CFTR) gene that result in premature stop codons (PTCs) in the messenger RNA (mRNA) generating truncated CFTR protein responsible for a severe CF phenotype. Pharmacological approaches have been proposed to directly overcome PTCs. Ataluren (PTC124) a small molecule that mimics the activity of aminoglycosides has been suggested to allow PTCs readthrough and to partially restore the protein function. However, des…

Adenosine negatively regulates duodenal motility in mice: role of A1 and A2A receptors

BACKGROUND AND PURPOSE Adenosine is considered to be an important modulator of intestinal motility. This study was undertaken to investigate the role of adenosine in the modulation of contractility in the mouse duodenum and to characterize the adenosine receptor subtypes involved. EXPERIMENTAL APPROACH RT-PCR was used to investigate the expression of mRNA encoding for A1, A2A, A2B and A3 receptors. Contractile activity was examined in vitro as changes in isometric tension. KEY RESULTS In mouse duodenum, all four classes of adenosine receptors were expressed, with the A2B receptor subtype being confined to the mucosal layer. Adenosine caused relaxation of mouse longitudinal duodenal muscle; …

RNA interference of MAD2 and BUBR1 genes causes mitotic spindle alterations, aneuploidy and cell cycle arrest p53-dependent.

The Spindle Assembly Checkpoint (SAC) is a cellular surveillance mechanism that functions to ensure faithful chromosome segregation during mitosis. Failure of this checkpoint or alteration in expression of SAC proteins (MAD2, BUB1, BUBR1 and others) can result in aneuploidy, a state of having abnormal numbers of chromosomes. MAD2 haploinsufficiency resulted in aneuploidy in MEFs and colon cancer cells in culture. Thus, spindle checkpoint components might have additional functions not-checkpoint-related functions that when disrupted contribute to tumorigenesis. Here we investigated the effects of MAD2 or BUBR1 transcriptional silencing in HCT-116 cells. Transient reduction of MAD2 (40%) and …

Evidence for region-specific effects of Glucagon-like Ppeptide-2 in mouse stomach”

Does the evidence matter in medicine? The retinoblastoma paradigm.

Retinoblastoma (Rb) is the most common intraocular malignant tumour in childhood, with an incidence of 1 in 15,000 live births. Complete information on this rare tumour can be easily accessed through the internet, although many aspect concerning the aetiology and pathogenesis of the disease, are still controversial. The "two hit" theory, formulated in 1971 to explain the variegated clinical expression of the disease, is based on the idea that single gene mutation may determine the development of cancer. However, this view does not take into account the most recent evidences showing the role of aneuploidy and chromosome instability in cancer. Also, a number of other genes and epigenetic mech…

Identification of mechanism(s) leading to hyperdiploidy in progenitor tumor cells derived from MCF7 breast cancer cells

Stem cells are a minor population of mostly resting cells defined by their long life, high clonogenicity, self-replicating potential, plasticity, and drug resistance (Finn, 2008). Cells with these properties have been identified in various normal and cancerous human tissues (Wicha, 2006), as well as in several long-term tumor cell lines (Setoguchi, 2004). We have some preliminary data indicating that cells isolated from MCF7 line divide slowly and form spheres, both features of progenitors tumor cells, when grown in ultralow adherent plates and in absence of serum. Furthermore, these features were associated to two distinct populations characterized by different content in terms of number o…

Novel Translational Read-through–Inducing Drugs as a Therapeutic Option for Shwachman-Diamond Syndrome

Shwachman-Diamond syndrome (SDS) is one of the most commonly inherited bone marrow failure syndromes (IBMFS). In SDS, bone marrow is hypocellular, with marked neutropenia. Moreover, SDS patients have a high risk of developing myelodysplastic syndrome (MDS), which in turn increases the risk of acute myeloid leukemia (AML) from an early age. Most SDS patients are heterozygous for the c.183-184TA>CT (K62X) SBDS nonsense mutation. Fortunately, a plethora of translational read-through inducing drugs (TRIDs) have been developed and tested for several rare inherited diseases due to nonsense mutations so far. The authors previously demonstrated that ataluren (PTC124) can restore full-length SBDS…

Investigating the inhibition of FTSJ1 a tryptophan tRNA-specific 2’-O-methyltransferase by NV TRIDs, as a mechanism of readthrough in nonsense mutated CFTR

Abstract: Cystic Fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the CFTR gene, coding for the CFTR chloride channel. About 10% of the CFTR gene mutations are "stop" mutations, which generate a Premature Termination Codon (PTC), thus synthesizing a truncated CFTR protein. A way to bypass PTC relies on ribosome readthrough, which is the ri-bosome’s capacity to skip a PTC, thus generating a full-length protein. “TRIDs” are molecules exerting ribosome readthrough; for some, the mechanism of action is still under debate. We in-vestigate a possible mechanism of action (MOA) by which our recently synthesized TRIDs, namely NV848, NV914, and NV930, could exert their r…

Transient and stable depletion of RB induce different expression of genes involved in epigenetic modifications.

ADENOSINE IS A MODULATOR OF THE CONTRACTILITY OF THE DUODENAL LONGITUDINAL MUSCLE IN MICE

DNA demethylation caused By 5-Aza-2'-Deoxycytidine induces mitotic alterations and aneuploidy

Aneuploidy, the unbalanced number of chromosomes in a cell, is considered a prevalent form of genetic instability and is largely acknowledged as a condition implicated in tumorigenesis. Epigenetic alterations like DNA hypomethylation have been correlated with cancer initiation/progression. Furthermore, a growing body of evidence suggests the involvement of epigenome-wide disruption as a cause of global DNA hypomethylation in aneuploidy generation. Here, we report that the DNA hypomethylating drug 5-aza-2′-deoxycytidine (DAC), affects the correct ploidy of nearly diploid HCT-116 human cells by altering the methylation pattern of the chromosomes. Specifically, we show that a DAC-induced reduc…

p14(ARF) Prevents Proliferation of Aneuploid Cells by Inducing p53-Dependent Apoptosis.

Weakening the Spindle Assembly Checkpoint by reduced expression of its components induces chromosome instability and aneuploidy that are hallmarks of cancer cells. The tumor suppressor p14(ARF) is overexpressed in response to oncogenic stimuli to stabilize p53 halting cell progression. Previously, we found that lack or reduced expression of p14(ARF) is involved in the maintenance of aneuploid cells in primary human cells, suggesting that it could be part of a pathway controlling their proliferation. To investigate this aspect further, p14(ARF) was ectopically expressed in HCT116 cells after depletion of the Spindle Assembly Checkpoint MAD2 protein that was used as a trigger for aneuploidy. …

Gastric relaxation induced by glucagon-like peptide-2 in mice fed a high-fat diet or fasted.

Glucagon-like peptide-2 (GLP-2) is a nutrient-responsive gut hormone that increases the intestinal absorption. Exogenous GLP-2 also induces gastric fundus relaxation with possible implications for emptying rate or feeling of satiety. GLP-2 actions are mediated by GLP-2 receptor (GLP-2R), located on enteric neurons and myofibroblasts in murine gastrointestinal tract. Because it is not known whether changes in the endogenous GLP-2R levels occur in different nutritional states, we examined the GLP-2R gene and protein expression in gastric fundus from standard diet (STD)-fed, 12-h and 24-h fasted and re-fed, or high-fat diet (HFD)-fed mice and we analyzed the mechanical responses to exogenous G…

The innovative role of the readthrough inducing drugs in the translation rescue of mRNAs characterized by premature stop codon (PTCs).

Simultaneous reduction of MAD2 and BUBR1 expression induces mitotic spindle alterations associated with p53 dependent cell cycle arrest and death

Most human tumors are characterized by aneuploidy that is believed to be the consequence of chromosomal instability (CIN). The mechanism(s) leading to aneuploidy and the pathways that allow its tolerance are not completely understood. The Spindle Assembly Checkpoint (SAC) is a cellular surveillance mechanism working during mitosis, and alterations of genes that encode components of the SAC weakening the mitotic checkpoint, induce aneuploidy by chromosome mis-segregation. We induced aneuploidy in near-diploid tumor cells by simultaneous depletion of the SAC proteins MAD2 and BUBR1 by RNA interference in the attempt to gain further insight on the cellular responses to aneuploidy. Individual r…

Valutazione dell'azione readthorough della molecola PTC124 su sistemi modello cellulari contenenti mutazioni non senso e in cellule epiteliali bronchiali IB3.1 (delF508/W1282X) derivate da pazienti affetti da fibrosi cistica.

Circa il 10% dei pazienti affetti da fibrosi cistica (FC) presenta nel gene CFTR mutazioni non senso (o 'stop': UGA, UAG or UAA, mutazioni di classe I) che bloccano prematuramente la sintesi della proteina. Attualmente non esiste una cura per questo tipo di mutazioni ma si sta cercando di individuare delle molecole che siano in grado di indurre la traduzione di codoni di stop prematuri (readthrough) che, rispetto a molecole già note come il G418, abbiano effetti collaterali ridotti ed una maggiore specificità per uno specifico codone. Una piccola molecola che sembra possedere una tale attività è il PTC124 (Welch, 2007). Ad oggi però non c’è ancora un consenso generale sul meccanismo di azio…

Transient silencing of MAD2 induces mitotic abnormalities and p21waf1 overexpression in primary human fibroblasts

Inhibitory control of duodenal contractility by adenosine in mice

Rescuing CFTR Protein Function: 1,3,4-oxadiazoles versus 1,2,4-oxadiazoles as readthrough inducing drugs

In Cystic fibrosis (CF) disease nonsense mutations in the CFTR gene cause the absence of the CFTR protein expression and a more severe form of the disease. About 10% of patient affected by CF show a nonsense mutation. A potential treatment of this alteration is to promote translational readthrough of premature termination codons (PTCs) by translational readthrough inducing drugs such as Ataluren (1). We reported a rationale for Ataluren promoted readthrough of PTCs by computational approach and GFP-reporter cell-based assay (2) and the observed enhancement of readthrough activity by some Ataluren derivatives (3, 4). In this context we aimed to compare the 1,2,4-oxadiazole core of Ataluren w…

Hydroxiurea induces centrosome amplification and slowing down of cell cycle in colo-cancer cells SW480

Micronuclei generation and chromosomal instability after RB interferences in human fibroblasts

Identification of pathways involved in aneuploidy onset and its tolerance using a DNA microarray approach

Genomic instability is a hallmark of the majority of human tumors explaining the heterogeneity shown by tumor cells. This phenomenon is often associated with chromosomal instability (CIN) and aneuploidy, a condition in which tumor cells lose or gain chromosomes. Previously, we showed that posttranscriptional silencing by RNAi of pRb1, DNMT12 and MAD2 is associated with aneuploidy in cultured human cells reinforcing the idea that there are several roads leading to aneuploidy. In the attempt to understand if a common molecular signature exists underlying aneuploidy and its tolerance in tumor cells, we induced aneuploidy in human fibroblasts (IMR90) by depleting Rb, MAD2 and DNMT1 genes and an…

Acute loss of pRB function induces centrosome amplification and aneuploidy both in murine and human fibroblasts

Translational readthrough inducing drugs: a study of toxicity in mice models and in vitro safety validation of the specific readthrough process.

Objective Nonsense mutations are responsible for 15% of Cystic Fibrosis (CF) patients due to the introduction of a premature stop codon (PTC) in the mRNA and the production of a truncated CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) protein1. A promising therapeutic approach for stop mutations is the suppression therapy by Translational Readthrough Inducing Drugs (TRIDs) to restore the expression of the protein2,3. Recently three new TRIDS (NV848, NV914, NV930) have been proposed and validated by several assays. Our work was focused on TRIDs NV848, NV914, NV930. Important aspects of TRIDs to be evaluated are their specificity towards PTC, to demonstrate that TRIDs do not inter…

RB acute loss induces centrosome amplification and aneuploidy in murine primary fibroblasts

AbstractBackgroundIncorrect segregation of whole chromosomes or parts of chromosome leads to aneuploidy commonly observed in cancer. The correct centrosome duplication, assuring assembly of a bipolar mitotic spindle, is essential for chromosome segregation fidelity and preventing aneuploidy. Alteration of p53 and pRb functions by expression of HPV16-E6 and E7 oncoproteins has been associated with centrosome amplification. However, these last findings could be the result of targeting cellular proteins in addition to pRb by HPV16-E7 oncoprotein. To get a more detailed picture on the role of pRb in chromosomal instability and centrosome amplification, we analyzed the effects of the acute loss …

pRb loss and chromosomal instability in human cells.

pRb loss and chromosomal instability in human cells. Recent studies suggest that Retinoblastoma tumor suppressor (RB) plays important roles in the prevention of chromosomal instability by regulating genes that control cell cycle progression and mitotic events. We investigated the effects of stable post-transcriptional silencing of RB in primary human fibroblasts (IMR90) and in near-diploid colon cancer cells (HCT116) focusing on chromosome missegregation mechanisms. Stable depletion of pRb was achieved by infection with the retroviral vector MSCV-LMP670 encoding a microRNA (miR670) targeting RB transcript. Cytogenetic, immunofluorescence microscopy and time-lapse video-microscopy analyses s…

Aneuploidy induced by MAD2 haploinsufficiency triggers premature senescence in human primary fibroblast

Aurora A overexpression leads centrosome amplification and aneuploidy in human colon cancer cells with MIN phenotype

Derivati Ossadiazolici per il trattamento della fibrosi cistica: Readthrough di mutazioni nonsense

Adenosine negatively regulates duodenal motility in mice: role of A1 and A2a receptors .

Investigating REPAIRv2 as a Tool to Edit CFTR mRNA with Premature Stop Codons

Cystic fibrosis (CF) is caused by mutations in the gene encoding the transmembrane conductance regulator (CFTR) protein. Some CF patients are compound heterozygous or homozygous for nonsense mutations in the CFTR gene. This implies the presence in the transcript of premature termination codons (PTCs) responsible for a truncated CFTR protein and a more severe form of the disease. Aminoglycoside and PTC124 derivatives have been used for the read-through of PTCs to restore the full-length CFTR protein. However, in a precision medicine framework, the CRISPR/dCas13b-based molecular tool &ldquo

Identification and validation of novel molecules obtained by integrated computational and experimental approaches for the readthrough of PTCs in CF cells

Cystic Fibrosis patients with nonsense-mutation in h-CFTR gene generally make virtually no CFTR protein and thus often have a more severe form of CF. Ataluren (PTC124) was suggested to induce read-through of premature but not normal termination codons. Despite the promising results there is not a general consensus on the mechanism of its action (protein stabilization or codon read-through) and its efficacy, the identification of new PTC124 analogues and the study of the mechanism of action may led to a new strategy for the development of a pharmacologic approach to the cure of CF.

Low doses of Hydroxyurea induce centrosome amplification and aneuploidy in primary human fibroblasts in culture

Strategies against nonsense: oxadiazoles as translational readthrough-inducing drugs (TRIDs)

This review focuses on the use of oxadiazoles as translational readthrough-inducing drugs (TRIDs) to rescue the functional full-length protein expression in mendelian genetic diseases caused by nonsense mutations. These mutations in specific genes generate premature termination codons (PTCs) responsible for the translation of truncated proteins. After a brief introduction on nonsense mutations and their pathological effects, the features of various classes of TRIDs will be described discussing differences or similarities in their mechanisms of action. Strategies to correct the PTCs will be presented, particularly focusing on a new class of Ataluren-like oxadiazole derivatives in comparison …

Aurora-A Transcriptional Silencing and Vincristine Treatment Show a Synergistic Effect in Human Tumor Cells

Aurora-A is a centrosome-associated serine/threonine kinase that is overexpressed in multiple types of human tumors. Primarily, Aurora-A functions in centrosome maturation and mitotic spindle assembly. Overexpression of Aurora-A induces centrosome amplification and G 2 /M cell cycle progression. Recently, it was observed that overexpression of Aurora-A renders cells resistant to cisplatin (CDDP)-, etoposide-, and paclitaxel-induced apoptosis.Our results indicate that already in initial stages of cancer progression Aurora-A overexpression could have a major role in inducing supernumerary centrosomes and aneuploidy, as shown by immunohistochemistry on tissue sections from various stages of hu…

Evidence for region-specific effects of glucagone-like peptide-2 in mouse stomach

Rescuing the CFTR protein function: Introducing 1,3,4-oxadiazoles as translational readthrough inducing drugs.

Nonsense mutations in the CFTR gene prematurely terminate translation of the CFTR mRNA leading to the production of a truncated protein that lacks normal function causing a more severe form of the cystic fibrosis (CF) disease. About 10% of patients affected by CF show a nonsense mutation. A potential treatment of this alteration is to promote translational readthrough of premature termination codons (PTCs) by Translational Readthrough Inducing Drugs (TRIDs) such as PTC124. In this context we aimed to compare the activity of PTC124 with analogues differing in the heteroatoms position in the central heterocyclic core. By a validated protocol consisting of computational screening, synthesis an…

Effects of Plk1 depletion on centrosome duplication and cell cycle progression in HCT116 tumor cells

STK15 overexpression in HCT116 cells causes mitotic disorders and hypodiploidy associated with centrosome amplification.

Uno studio comparativo in silico sui possibili target di Ataluren e analoghi farmaci promotori di readthrough di codoni di stop prematuri

E’ noto in letteratura che Ataluren (acido 5-(fluorofenil)-1,2,4-ossadiazolil-benzoico) sia in grado di sopprimere le mutazioni non senso favorendo il readthrough dei codoni di stop prematuri, anche se il suo meccanismo di azione non risulta ancora chiaro. La probabile interazione tra Ataluren e CTFR-mRNA è stata precedentemente studiata mediante dinamica molecolare. In questo studio1, abbiamo esteso il modeling del probabile meccanismo di azione di Ataluren mediante approcci computazionali completementari, quali Induced Fit Docking (IFD), Quantum Polarized Ligand Docking (QPLD), metodi MM-GBSA e mutagenesi computazionale. Oltre a considerare il CTFR-mRNA, sono stati presi in considerazione…

AURKA (aurora kinase A)

Review on AURKA (aurora kinase A), with data on DNA, on the protein encoded, and where the gene is implicated.

Aging modifies receptor expression but not muscular contractile response to angiotensin II in rat jejunum

AbstractThe involvement of renin-angiotensin system in the modulation of gut motility and age-related changes in mRNA expression of angiotensin (Ang II) receptors (ATR) are well accepted. We aimed to characterize, in vitro, the contractile responses induced by Ang II, in jejunum from young (3–6 weeks old) and old rats (≥ 1 year old), to evaluate possible functional differences associated to changes in receptor expression. Mechanical responses to Ang II were examined in vitro as changes in isometric tension. ATR expression was assessed by qRT-PCR. Ang II induced a contractile effect, antagonized by losartan, AT1R antagonist, and increased by PD123319, AT2R antagonist, as well by neural block…

Rb depletion induces centrosome amplification and different exression of mitotic genes in HCT116 tumor cells

Pharmacophore-Based Design of New Chemical Scaffolds as Translational Readthrough-Inducing Drugs (TRIDs)

[Image: see text] Translational readthrough-inducing drugs (TRIDs) rescue the functional full-length protein expression in genetic diseases, such as cystic fibrosis, caused by premature termination codons (PTCs). Small molecules have been developed as TRIDs to trick the ribosomal machinery during recognition of the PTC. Herein we report a computational study to identify new TRID scaffolds. A pharmacophore approach was carried out on compounds that showed readthrough activity. The pharmacophore model applied to screen different libraries containing more than 87000 compounds identified four hit-compounds presenting scaffolds with diversity from the oxadiazole lead. These compounds have been s…

Inhibition of FTSJ1, a tryptophan tRNA-specific 2’-O-methyltransferase as possible mechanism to readthrough premature termination codons (UGAs) of the CFTR mRNA

Cystic Fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the CFTR gene, coding for the CFTR chloride channel. About 10 % of the mutations affecting the CFTR gene are "stop" mutations, which generate a Premature Termination Codon (PTC), thus resulting in the synthesis of a truncated CFTR protein. A way to bypass PTC relies on ribosome readthrough, that is the capacity of the ribosome to skip a PTC, thus generating a full-length protein. “TRIDs” are molecules exerting ribosome readthrough and for some of them the mechanism of action is still under debate. By in silico analysis as well as in vitro studies, we investigate a possible mechanism of action (MOA) by whic…

Glucagon-like peptide-2 analog and inflammatory state in obese mice

Obesity is characterized by chronic low grade of systemic inflammation that develops in response to nutrient excess and plays a key role in the pathogenesis of insulin resistance. It is characterized by macrophage infiltration into adipose tissue (AT) and abnormal cytokine production. These factors damage the metabolic homeostasis leading to alteration in the insulin signaling in specific tissues and organs such as AT and liver. Thus, obese subjects develop over the time resistance to the cellular actions of insulin. Glucagon like peptide-2 (GLP-2) is an intestinal proglucagon-derived hormone released together with GLP1, in response to the passage of food by the distal small intestine. Once…

Inhalable nano into micro dry powders for ivacaftor delivery: The role of mannitol and cysteamine as mucus-active agents.

In this paper the innovative approach of Nano into micro (NiM9 was developed to produce Nanoparticles loaded Ivacaftor to incorporate into mannitol or mannitol/cysteamine micromatrices for drug pulmonary administration in CF. Nanoparticles composed by a mixture of two polyhydrohydroxyethtylaspartamide copolymers containing a loading of Ivacaftor of 15.5 % w/w were produced. These Nanoparticles were incorporated into microparticles to obtain NiM that were characterized in terms of size and size distribution, interaction with CF-AM by rheological and turbidimetric studies as well as by aerodynamic diameter measurements. Finally the activity of Ivacaftor into these NiM was evaluated by in vitr…

AZIONE READTHROUGH DI DERIVATI DEL PTC124 SU SISTEMI MODELLO CELLULARI E IN CELLULE DI EPITELIO BRONCHIALE-FC IB3.1 (CFTR F508/W1282X )

Obiettivi specifici: Le mutazioni nonsenso (mutazioni STOP), un difetto genetico frequente negli individui affetti da Fibrosi Cistica (CF), causano la sintesi di proteine CFTR tronche e non funzionanti che sono associate ad un fenotipo più severo della CF (McKone EF. et al., Chest 2006). L’obiettivo del nostro studio è stato quello di disegnare derivati dell’Ataluren (PTC124), una ‘small molecule’ a cui è stata attribuita attività readthrough, e valutarne l’attività su tre differenti sistemi modello sperimentali contenenti codoni di STOP prematuri (UGA, UAG, UAA). Materiali e metodi: Sono state sintetizzate 24 molecole derivate dal PTC124 e analizzate mediante tecniche spettroscopiche per v…

EFFECTS OF PLK1 DEPLETION IN CENTROSOME DUPLICATIONS AND CELL CYCLEPROGRESSION IN HCT116 TUMOR CELLS.

Nanopiattaforme di Ossido di Grafene perfluorurate: materiali biocompatibili con affinità per l'ossigeno

L’attacco covalente di molecule perfluorurate su ossido di graphene (GO) via sostituzione nucleofila aromatica ha consentito una facile funzionalizzazione delle lamelle di GO.1,2 La performance delle nanopiattaforme perluorurate (GOF) nell’uptake e rilascio di ossigeno è stata valutata in condizioni di pH fisiologico a differenti concentrazioni e temperature, dimostrando una maggiore affinità del materiale perfluorurato GOF rispetto al GO pristino. Anche a basse concentrazioni GOF ha mostrato valori di uptake di ossigeno a saturazione e velocità di diffusione superiori ad altri materiali proposti in letteratura per ingegneria tissutale, per l’ossigenazione cellulare durante la rigenerazione…

PTC124 DERIVATIVES AS A NOVEL APPROACH TO IMPROVE THE READTHROUGH OF PREMATURE AMBER AND OCHRE STOP CODONS

Nucleotide changes within an exon may alter the trinucleotide normally encoding a particular amino acid, such that a new “stop” signal is transcribed into the mRNA open reading frame. This causes the ribosome to prematurely terminate its reading of the mRNA, leading to the lack of production of a normal full-length protein. Such premature termination codon (PTC) mutations occur in an estimated 10% to 15% of many genetically based disorders (1). Pathological nonsense mutations resulting in TAG (40.4%), TGA (38.5%), and TAA (21.1%) occur in different proportions to naturally occurring stop codons (2). Several genetic disorders are characterized by opal (TGA; Cystic fibrosis, Duchenne/Becker m…

Perfluorocarbons–graphene oxide nanoplatforms as biocompatible oxygen reservoirs

Abstract 3-Pentadecafluoroheptyl,5-perfluorophenyl-1,2,4-oxadiazole (FOX) molecules were attached onto a graphene oxide (GO) via a facile aromatic substitution in alkaline environment. This approach allows achieving high degree of functionalization under mild conditions. The covalent attachment of perfluoromoieties onto GO lamellae was confirmed by spectroscopic analyses. The performance of these nanoplatforms (GOF) as oxygen reservoirs was assessed at different concentrations and temperature. The results revealed that under physiologic conditions GO and FOX synergistically operate for increasing oxygen uptake and release, either from a thermodynamic and a kinetic point of view. Even at low…

Effetti del silenziamento dei geni Plk1, Brca1 ed Rb nella duplicazione dei centrosomi in cellule tumorali HCT116

A1 ADENOSINE RECEPTOR MODULATION OF CONTRACTILITY IN MOUSE DUODENUM LONGITUDINAL MUSCLE.

Experimental evidence suggests that adenosine is involved in the regulation of gastrointestinal functions. In the present study we examined the influence of adenosine on the contractile activity of mouse duodenum longitudinal muscle. Reverse transcription-polymerase chain reaction revealed the expression of all the adenosine receptors in whole thickness duodenum, being the A2B receptors expressed only in the neuromuscular layer. Mechanical activity of longitudinally oriented duodenal segments was recorded in vitro as changes in isometric tension. Adenosine produced concentration-dependent relaxation, markedly reduced by DPCPX, A1 receptor antagonist, but unaffected by DMPX or MRS 1220, A2 a…

Functional Inactivation of pRB Results in Aneuploid Mammalian Cells After Release From a Mitotic Block

AbstractThe widespread chromosome instability observed in tumors and in early stage carcinomas suggests that aneuploidy could be a prerequisite for cellular transformation and tumor initiation. Defects in tumor suppressers and genes that are part of mitotic checkpoints are likely candidates for the aneuploid phenotype. By using flow cytometric, cytogenetic, immunocytochemistry techniques we investigated whether pRB deficiency could drive perpetual aneuploidy in normal human and mouse fibroblasts after mitotic checkpoint challenge by microtubule-destabilizing drugs. Both mouse and human pRB-deficient primary fibroblasts resulted, upon release from a mitotic block, in proliferating aneuploid …

X CONVENTION OF INVESTIGATORS IN CYSTIC FIBROSIS.

Background Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). Approximately 10% (worldwide) of patients have in-frame nonsense mutations (UAA, UAG or UGA class I mutations) in the CF trans-membrane regulator (CFTR) gene that result in premature stop codons (PTCs) in the messenger RNA (mRNA) generating truncated CFTR protein responsible for a severe CF phenotype. Pharmacological approaches have been proposed to directly overcome PTCs. Ataluren (PTC124) a small molecule that mimics the activity of aminoglycosides has been suggested to allow PTCs readthrough and to partially restore the protein function. However, des…

Exploring the readthrough of nonsense mutations by non-acidic Ataluren analogues selected by ligand-based virtual screening

Abstract Ataluren, also known as PTC124, is a 5-(fluorophenyl)-1,2,4-oxadiazolyl-benzoic acid suggested to suppress nonsense mutations by readthrough of premature stop codons in the mRNA. Potential interaction of PTC124 with mRNA has been recently studied by molecular dynamics simulations highlighting the importance of H-bonding and stacking π−π interactions. A series of non-acidic analogues of PTC124 were selected from a large database via a ligand-based virtual screening approach. Eight of them were synthesized and tested for their readthrough activity using the Fluc reporter harboring the UGA premature stop codon. The most active compound was further tested for suppression of the UGA non…

p14ARFPrevents Proliferation of Aneuploid Cells by Inducing p53-Dependent Apoptosis

Weakening the Spindle Assembly Checkpoint by reduced expression of its components induces chromosome instability and aneuploidy that are hallmarks of cancer cells. The tumor suppressor p14ARF is overexpressed in response to oncogenic stimuli to stabilize p53 halting cell progression. Previously, we found that lack or reduced expression of p14ARF is involved in the maintenance of aneuploid cells in primary human cells, suggesting that it could be part of a pathway controlling their proliferation. To investigate this aspect further, p14ARF was ectopically expressed in HCT116 cells after depletion of the Spindle Assembly Checkpoint MAD2 protein that was used as a trigger for aneuploidy. p14ARF…

Silencing of serine threonine kinase Aurora-2 gene causes G2/M arrest and apoptosis in human carcinoma lung large cells.

DNMT1 depletion activates a pathway p14ARF/TP53 controlled that induces G1 arrest preventing DNA demethylation and aneuploidy

Background: Aneuploidy is considered the result of chromosome segregation errors caused by defects in the mitotic spindle assembly, centrosome duplication, cell-cycle checkpoints and epigenetic changes. Usually, aneuploidy affects negatively proliferation of normal cells. However, it is frequently associated with cancer that is characterized by a uncontrolled proliferation. Thus, understanding the pathway(s) that block proliferation of aneuploid cells might open new avenue to exploit new cancer therapies. O bservations: We found that in primary human fi broblasts (IMR90) knocking down of DNMT1, a member of epigenetic machinery is perceived by the cell as a stress signal that induces p14ARF …

OXADIAZOLE DERIVATIVES FOR THE TREATMENT OF GENETIC DISEASES DUE TO NONSENSE MUTATIONS

Are disclosed oxadiazole derivatives, their use as medicaments and in particular for the treatment of diseases associated with the presence of a nonsense mutation in the gene or a premature stop codon in the mRNA, pharmaceutical formulation comprising said oxadiazole derivatives and prodrug or mixture thereof and the methods for the preparation of said Oxadiazole derivatives.

Silencing of serine threonine kinase Aurora-2 gene cause G2/M arrest and apotosis in human carcinoma lung large cells

Readthrough Inducing Drugs (TRIDs) in human fibroblasts harboring the c.5047 C>T (R1683*) nonsense mutation

Missing Evidences in Cancer Genetics: The Retinoblastoma Paradigm

BACKGROUND: Retinoblastoma (Rb) is the most common primary malignant intraocular tumour in childhood. The "two hit" theory, formulated by Knudson in 1971 to explain the variegated clinical expression of the disease, led to the discovery of the so called tumour suppressor genes and the identification of the Rb1 as the prototype of such genes. Mutations of the Rb1 gene are now commonly believed to be the "cause" retinoblastoma, although epidemiological, clinical, and biological evidences argue against it. MATERIAL/METHODS: The Authors have performed a systematic review of available data concerning clinical and diagnostic aspects of retinoblastoma, including molecular genetics. Meta analysis o…

TRPM8 Channel Activation Reduces the Spontaneous Contractions in Human Distal Colon

The transient receptor potential-melastatin 8 (TRPM8) is a non-selective Ca2+-permeable channel, activated by cold, membrane depolarization, and different cooling compounds. TRPM8 expression has been found in gut mucosal, submucosal, and muscular nerve endings. Although TRPM8 plays a role in pathological conditions, being involved in visceral pain and inflammation, the physiological functions in the digestive system remain unclear as yet. The aims of the present study were: (i) to verify the TRPM8 expression in human distal colon

Simultaneous reduction of MAD2 and BUBR1 expression induces mitotic spindle alterations associated with p53 dependent cell cycle arrest and death

Toward a Rationale for the PTC124 (Ataluren) Promoted Readthrough of Premature Stop Codons: A Computational Approach and GFP-Reporter Cell-Based Assay

The presence in the mRNA of premature stop codons (PTCs) results in protein truncation responsible for several inherited (genetic) diseases. A well-known example of these diseases is cystic fibrosis (CF), where approximately 10% (worldwide) of patients have nonsense mutations in the CF transmembrane regulator (CFTR) gene. PTC124 (3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)-benzoic acid), also known as Ataluren, is a small molecule that has been suggested to allow PTC readthrough even though its target has yet to be identified. In the lack of a general consensus about its mechanism of action, we experimentally tested the ability of PTC124 to promote the readthrough of premature termination c…

Bypass of cell cycle arrest induced by transient DNMT1 post-transcriptional silencing triggers aneuploidy in human cells

Abstract Background Aneuploidy has been acknowledged as a major source of genomic instability in cancer, and it is often considered the result of chromosome segregation errors including those caused by defects in genes controlling the mitotic spindle assembly, centrosome duplication and cell-cycle checkpoints. Aneuploidy and chromosomal instability has been also correlated with epigenetic alteration, however the molecular basis of this correlation is poorly understood. Results To address the functional connection existing between epigenetic changes and aneuploidy, we used RNA-interference to silence the DNMT1 gene, encoding for a highly conserved member of the DNA methyl-transferases. DNMT1…

CENPA overexpression promotes genome instability in pRb-depleted human cells

Abstract Background Aneuploidy is a hallmark of most human cancers that arises as a consequence of chromosomal instability and it is frequently associated with centrosome amplification. Functional inactivation of the Retinoblastoma protein (pRb) has been indicated as a cause promoting chromosomal instability as well centrosome amplification. However, the underlying molecular mechanism still remains to be clarified. Results Here we show that pRb depletion both in wild type and p53 knockout HCT116 cells was associated with the presence of multipolar spindles, anaphase bridges, lagging chromosomes and micronuclei harbouring whole chromosomes. In addition aneuploidy caused by pRb acute loss was…

Glucagon-like peptide-1 relaxes gastric antrum through nitric oxide in mice.

Abstract Glucagon-like-peptide-1 (GLP-1) is a proglucagon-derived peptide expressed in the intestinal enteroendocrine-L cells and released after meal ingestion. GLP-1 reduces postprandial glycemia not only by its hormonal effects, but also by its inhibitory effects on gastrointestinal motility. Recently, we showed that GLP-1 acts in the enteric nervous system of mouse intestine. Therefore our working hypothesis was that GLP-1 may have also a direct influence on the gastric mechanical activity since the major part of experimental studies about its involvement in the regulation of gastric motility have been conducted in in vivo conditions. The purposes of this study were (i) to examine exogen…

DNMT1 transient silencing induces aneuploidy, premature separated chromatids and centromeric chromatin alterations

Targeting Nonsense: Optimization of 1,2,4-Oxadiazole TRIDs to Rescue CFTR Expression and Functionality in Cystic Fibrosis Cell Model Systems

Cystic fibrosis (CF) patients develop a severe form of the disease when the cystic fibrosis transmembrane conductance regulator (CFTR) gene is affected by nonsense mutations. Nonsense mutations are responsible for the presence of a premature termination codon (PTC) in the mRNA, creating a lack of functional protein. In this context, translational readthrough-inducing drugs (TRIDs) represent a promising approach to correct the basic defect caused by PTCs. By using computational optimization and biological screening, we identified three new small molecules showing high readthrough activity. The activity of these compounds has been verified by evaluating CFTR expression and functionality after…

“DNA Methyl transferase 1 post-trascriptional silencing indues aneuploidy and cell cycle arrest in human cells”,

Aurora-A/STK15 transcriptional silencing and Vincristine treatment show a synergistic effect in human tumour cells