Domenica Carnicelli

Novel Dyskerin-Mediated Mechanism of p53 Inactivation through Defective mRNA Translation

In up to 60% of human cancers, p53 gene mutations are responsible for direct inactivation of the tumor suppressor function of p53. Alternative mechanisms of p53 inactivation described thus far mainly affect its posttranslational regulation. In X-linked dyskeratosis congenita, a multisystemic syndrome characterized by increased cancer susceptibility, mutations of the DKC1 gene encoding dyskerin cause a selective defect in the translation of a subgroup of internal ribosome entry site (IRES)-containing cellular mRNAs. In this study, we show that impairment of dyskerin function can cause p53 inactivation due to a defect in p53 mRNA translation. siRNA-mediated reduction of dyskerin levels caused a decrease of p53 mRNA translation, protein levels, and functional activity, both in human breast cancer cells and in primary mammary epithelial progenitor cells. These effects seemed to be independent of the known role of dyskerin in telomerase function, and they were associated with a specific impairment of translation initiation mediated by IRES elements present in p53 mRNA. In a series of human primary breast cancers retaining wild-type p53, we found that low levels of dyskerin expression were associated with reduced expression of p53-positive target genes. Our findings suggest that a dyskerin-mediated mechanism of p53 inactivation may occur in a subset of human tumors.

Purification and properties of new ribosome-inactivating proteins with RNA N-glycosidase activity

Ribosome-inactivating proteins (RIPs) similar to those already known (Stirpe & Barbieri (1986) FEBS Lett. 195, 1-8) were purified from the seeds of Asparagus officinalis (two proteins, asparin 1 and 2), of Citrullus colocynthis (two proteins, colocin 1 and 2), of Lychnis chalcedonica (lychnin) and of Manihot palmata (mapalmin), from the roots of Phytolacca americana (pokeweed antiviral protein from roots, PAP-R) and from the leaves of Bryonia dioica (bryodin-L). The two latter proteins can be considered as isoforms, respectively, of previously purified PAP, from the leaves of P. americana, and of bryodin-R, from the roots of B. dioica. All proteins have an Mr at approx, 30,000, and an alkaline isoelectric point. Bryodin-L, colocins, lychnin and mapalmin are glycoproteins. All RIPs inhibit protein synthesis by a rabbit reticulocyte lysate and phenylalanine polymerization by isolated ribosomes and alter rRNA in a similar manner as the A-chain of ricin and related toxins (Endo et al. (1987) J. Biol. Chem. 262, 5908-5912).

Effects of osmolarity, ions and compatible osmolytes on cell-free protein synthesis.

To mimic what might happen in cells exposed to hypertonicity, the effects of increased osmolarity and ionic strength on cell-free protein synthesis have been examined. Translation of globin mRNA by rabbit reticulocyte lysate decreased by 30-60% when osmolality was increased from 0.35 to 0.53 osmol/kg of water by the addition of NaCl, KCl, CH(3)CO(2)Na or CH(3)CO(2)K. In contrast, equivalent additions of the compatible osmolytes betaine or myo -inositol caused a 40-50% increase in the rate of translation, whereas amino acids (50-135 mM) that are transported via system A had no significant effect. Addition of 75 mM KCl caused a dramatic fall in the amount of the 43 S pre-initiation complex, whereas it was totally preserved when osmolarity was similarly increased by the addition of 150 mM betaine. The formation of a non-enzymic initiation complex between rabbit [(3)H]Phe-tRNA, poly(U) and the 80 S ribosomes was unaffected by the addition of 75 mM NaCl or KCl, but translation of the complex decreased by 70%. Density-gradient centrifugation of reticulocyte extracts translating endogenous mRNA revealed that addition of 150 mM betaine had no effect, whereas addition of 75 mM KCl caused a marked decrease in the polysome peak, concomitant with an increase in the proportion of 80 S ribosomes and ribosomal subunits, even when elongation was inhibited with fragment A of diphtheria toxin. These results are consistent with the notion that both initiation and elongation are inhibited by unusually high concentrations of inorganic ions, but not by the compatible osmolytes betaine or myo -inositol.

Clinical relevance of shiga toxin concentrations in the blood of patients with hemolytic uremic syndrome

Background: Intestinal infections with Shiga toxin-producing Escherichia coli (STEC) in children can lead to the hemolytic uremic syndrome (HUS). Shiga toxins (Stx) released in the gut by bacteria enter the blood stream and target the kidney causing endothelial injury. Free toxins have never been detected in the blood of HUS patients, but they have been found on the surface of polymorphonuclear leukocytes (PMN). Methods: With respect to their clinical features, the clinical relevance of the amounts of serum Stx (cytotoxicity assay with human endothelial cells) and PMN-bound Stx (cytofluorimetric assay) in 46 patients with STEC-associated HUS was evaluated. Results: Stx-positive PMN were found in 60% of patients, whereas negligible amounts of free Stx were detected in the sera. Patients with high amounts of Stx on PMN showed preserved or slightly impaired renal function (incomplete form of HUS), whereas cases with low amounts of Stx usually presented evidence of acute renal failure. Conclusions: These observations suggest that the extent of renal damage in children with STEC-associated HUS could depend on the concentration of Stx present on their PMN and presumably delivered by them to the kidney. As previously shown by experimental models from our laboratory, high amounts of Stx could induce a reduced release of cytokines by the renal endothelium, with a consequent lower degree of inflammation. Conversely, low toxin amounts can trigger the cytokine cascade, provoking inflammation, thereby leading to tissue damage.

Interactions between Shiga toxins and human polymorphonuclear leukocytes

Human intestinal infections by Shiga toxin (Stx)‐producing Escherichia coli cause hemorrhagic colitis and hemolytic uremic syndrome (HUS), which represents the main cause of acute renal failure in early childhood. In HUS, Stx released in the gut enter the bloodstream and are targeted to renal endothelium. The mechanism of toxin delivery is still a matter of debate, although the role of polymorphonuclear leukocytes (PMN) as a Stx carrier has been indicated. The aim of this paper was to better define the interactions between Stx and human PMN. Direct and indirect flow cytometric analysis and binding experiments with radiolabeled toxins demonstrated that Stx bind to the surface of human mature PMN but not to immature PMN from G‐CSF‐treated donors. The use of the human myeloid leukemia cell (HL‐60) model for inducible cell differentiation confirmed that the toxin binding occurs only after granulocytic differentiation. Stx binding caused a delay of the spontaneous apoptosis of PMN, as shown by the delayed appearance of apoptotic nuclei and activation of caspase 3 and by the higher number of cells negative to the annexin V‐binding assay after 48 h. Moreover, flow cytometric analysis of mixed Stx‐positive and Stx‐negative PMN populations showed that the toxins were transferred from positive to negative PMN. The delayed, spontaneous apoptosis and the passage of the toxic ligand from older PMN to new, mature cells entering the circulation from the bone marrow may explain the previously reported persistence of Stx in the blood of children with HUS.

Creatine as a compatible osmolyte in muscle cells exposed to hypertonic stress.

Exposure of C2C12 muscle cells to hypertonic stress induced an increase in cell content of creatine transporter mRNA and of creatine transport activity, which peaked after about 24 h incubation at 0.45 osmol (kg H2O)−1. This induction of transport activity was prevented by addition of either cycloheximide, to inhibit protein synthesis, or of actinomycin D, to inhibit RNA synthesis. Creatine uptake by these cells is largely Na+ dependent and kinetic analysis revealed that its increase under hypertonic conditions resulted from an increase in Vmax of the Na+‐dependent component, with no significant change in the Km value of about 75 μmol l−1. Quantitative real‐time PCR revealed a more than threefold increase in the expression of creatine transporter mRNA in cells exposed to hypertonicity. Creatine supplementation significantly enhanced survival of C2C12 cells incubated under hypertonic conditions and its effect was similar to that obtained with the well known compatible osmolytes, betaine, taurine and myo‐inositol. This effect seemed not to be linked to the energy status of the C2C12 cells because hypertonic incubation caused a decrease in their ATP content, with or without the addition of creatine at 20 mmol l−1 to the medium. This induction of creatine transport activity by hypertonicity is not confined to muscle cells: a similar induction was shown in porcine endothelial cells.

Flow cytometry detection of Shiga toxins in the blood from children with hemolytic uremic syndrome.

Hemolytic uremic syndrome (HUS) is the main cause of acute renal failure in early childhood. Most cases are due to intestinal infections from Escherichia coli strains (STEC) which produce by Shiga toxin (Stxs). Stx1 and Stx2 produced by STEC in the gut are absorbed into the circulation and, after binding on polymorphonuclear leukocytes (PMNs), are targeted to renal endothelium. The aim of the present work was the development of a method to detect Stxs bound on circulating PMNs and to diagnose STEC infections in patients with HUS.

Ribosome-inactivating proteins depurinate poly(ADP-ribosyl)ated poly(ADP-ribose) polymerase and have transforming activity for 3T3 fibroblasts

It has been known that ribosome‐inactivating proteins (RIPs) from plants damage ribosomes by removing adenine from a precise position of rRNA. Subsequently it was observed that all tested RIPs depurinate DNA, and some of them also non‐ribosomal RNAs and poly(A), hence the denomination of adenine polynucleotide glycosylases was proposed. We report now that ricin, saporin‐L2, saporin‐S6, gelonin and momordin depurinate also poly(ADP‐ribosyl)ated poly(ADP‐ribose) polymerase (auto modified enzyme), an enzyme involved in DNA repair. We observed also that all RIPs but gelonin induce transformation of fibroblasts, possibly as a consequence of damage to DNA and of the altered DNA repair system.

Shiga Toxins Present in the Gut and in the Polymorphonuclear Leukocytes Circulating in the Blood of Children with Hemolytic-Uremic Syndrome

ABSTRACT Hemolytic-uremic syndrome, the main cause of acute renal failure in early childhood, is caused primarily by intestinal infections from some Escherichia coli strains that produce Shiga toxins. The toxins released in the gut are targeted to renal endothelium after binding to polymorphonuclear leukocytes. The presence of Shiga toxins in the feces and the circulating neutrophils of 20 children with hemolytic uremic syndrome was evaluated by the Vero cell cytotoxicity assay and flow cytometric analysis, respectively. The latter showed the presence of Shiga toxins on the polymorphonuclear leukocytes of 13 patients, 5 of whom had no other microbiologic or serologic evidence of infection by Shiga toxin-producing Escherichia coli. A positive relationship was observed between the amounts of Shiga toxins released in the intestinal lumen and those released in the bloodstream. The toxins were detectable on the neutrophils for a median period of 5 days after they were no longer detectable in stools. This investigation confirms that the immunodetection of Shiga toxins on neutrophils is a valuable tool for laboratory diagnosis of Shiga toxin-producing Escherichia coli infection in hemolytic-uremic syndrome and provides clues for further studies on the role of neutrophils in the pathogenesis of this syndrome.

Identification of TLR4 as the Receptor That Recognizes Shiga Toxins in Human Neutrophils

Hemolytic uremic syndrome (HUS) caused by intestinal Shiga toxin–producing Escherichia coli infections is a worldwide health problem, as dramatically exemplified by the German outbreak occurred in summer 2011 and by a constant burden of cases in children. Shiga toxins (Stx) play a pivotal role in HUS by triggering endothelial damage in kidney and brain through globotriaosylceramide (Gb3Cer) receptor targeting. Moreover, Stx interact with human neutrophils, as experimentally demonstrated in vitro and as observed in patients with HUS. A neutrophil-protective role on endothelial damage (sequestration of circulating toxins) and a causative role in toxin delivery from the gut to the kidney (piggyback transport) have been suggested in different studies. However, the receptor that recognizes Stx in human neutrophils, which do not express Gb3Cer, has not been identified. In this study, by competition and functional experiments with appropriate agonists and antagonists (LPS, anti-TLR4 Abs, respectively), we have identified TLR4 as the receptor that specifically recognizes Stx1 and Stx2 in human neutrophils. Accordingly, these treatments displaced both toxin variants from neutrophils and, upon challenge with Stx1 or Stx2, neutrophils displayed the same pattern of cytokine expression as in response to LPS (assessed by quantitative RT-PCR, ELISA, or multiplexed Luminex-based immunoassays). Moreover, data were supported by adequate controls excluding any potential interference of contaminating LPS in Stx-binding and activation of neutrophils. The identification of the Stx-receptor on neutrophils provides additional elements to foster the understanding of the pathophysiology of HUS and could have an important effect on the development of therapeutic strategies.