Expression of bovine leukemia virus ENV glycoprotein in insect cells by recombinant baculovirus

AbstractThe gp51-p30 glycoprotein constituting BLV envelope was expressed in Sf-21 insect cells by means of recombinant baculoviruses. Post-infection cell lysates were analyzed, in order to define the immunologic reactivity of recombinant products. Oligosaccharide chains, containing N-acetylglucosamine, mannose, galactose and sialic acid were found on recombinant gp51-p30. In order to investigate the timing of transcription and translation of the glycoprotein, kinetic assays were carried out on cell lysates and directly in situ on Sf-21 cells during the course of baculovirus infection. The use of different solubilizing reagents was also evaluated in order to rescue recombinant glycoprotein from its subcellular location

Replication in mammalian cells recapitulates the locus-specific differences in somatic instability of genomic GAA triplet-repeats

Friedreich ataxia is caused by an expanded (GAA·TTC)(n) sequence in intron 1 of the FXN gene. Small pool PCR analysis showed that pure (GAA·TTC)(44+) sequences at the FXN locus are unstable in somatic cells in vivo, displaying both expansions and contractions. On searching the entire human and mouse genomes we identified three other genomic loci with pure (GAA·TTC)(44+) sequences. Alleles at these loci showed mutation loads of <1% compared with 6.3–30% for FXN alleles of similar length, indicating that somatic instability in vivo is regulated by locus-specific factors. Since distance between the origin of replication and the (CTG·CAG)(n) sequence modulates repeat instability in mammalian cells, we tested if this could also recapitulate the locus-specific differences for genomic (GAA·TTC)(n) sequences. Repeat instability was evaluated following replication of a (GAA·TTC)(115) sequence in transfected COS1 cells under the control of the SV40 origin of replication located at one of five different distances from the repeat. Indeed, depending on the location of the SV40 origin relative to the (GAA·TTC)(n) sequence, we noted either no instability, predominant expansion or both expansion and contraction. These data suggest that mammalian DNA replication is a possible mechanism underlying locus-specific differences in instability of GAA triplet-repeat sequences

Chemotherapy elicits pro-metastatic extracellular vesicles in breast cancer models

Cytotoxic chemotherapy is an effective treatment for invasive breast cancer. However, experimental studies in mice also suggest that chemotherapy has pro-metastatic effects. Primary tumours release extracellular vesicles (EVs), including exosomes, that can facilitate the seeding and growth of metastatic cancer cells in distant organs, but the effects of chemotherapy on tumour-derived EVs remain unclear. Here we show that two classes of cytotoxic drugs broadly employed in pre-operative (neoadjuvant) breast cancer therapy, taxanes and anthracyclines, elicit tumour-derived EVs with enhanced pro-metastatic capacity. Chemotherapy-elicited EVs are enriched in annexin A6 (ANXA6), a Ca2+-dependent protein that promotes NF-κB-dependent endothelial cell activation, Ccl2 induction and Ly6C+CCR2+ monocyte expansion in the pulmonary pre-metastatic niche to facilitate the establishment of lung metastasis. Genetic inactivation of Anxa6 in cancer cells or Ccr2 in host cells blunts the prometastatic effects of chemotherapy-elicited EVs. ANXA6 is detected, and potentially enriched, in the circulating EVs of breast cancer patients undergoing neoadjuvant chemotherapy

Strategie usate per localizzare geni all'interno di genomi complessi

Dottorato di ricerca in biotecnologie applicate alle scienze veterinarie e zootecniche. 8. ciclo. Coordinatore e docente guida G. Poli. Controrelatori L. Ferretti e G. FinocchiaroConsiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome; Biblioteca Nazionale Centrale - P.za Cavalleggeri, 1, Florence / CNR - Consiglio Nazionale delle RichercheSIGLEITItal

Oncogenic ras-driven cancer cell vesiculation leads to emission of doublestranded DNA capable of interacting with target cells

Cell free DNA is often regarded as a source of genetic cancer biomarkers, but the relatedmechanisms of DNA release, composition and biological activity remain unclear. Here we showthat rat epithelial cell transformation by the human H-ras oncogene leads to an increase inproduction of small, exosomal-like extracellular vesicles by viable cancer cells. These EVscontain chromatin-associated double-stranded DNA fragments covering the entire host genome,including full-length H-ras. Oncogenic N-ras and SV40LT sequences were also found in EVsemitted from spontaneous mouse brain tumour cells. Disruption of acidic sphingomyelinase andthe p53/Rb pathway did not block emission of EV-related oncogenic DNA. Exposure of nontransformedRAT-1 cells to EVs containing mutant H-ras DNA led to the uptake and retention ofthis material for an extended (30 days) but transient period of time, and stimulated cellproliferation. Thus, our study suggests that H-ras-mediated transformation stimulates vesicularemission of this histone-bound oncogene, which may interact with non-transformed cells

Impact of host ageing on the metastatic phenotype

Ageing impacts multiple host mechanisms involved in cancer progression. Here we show that poorly metastatic Lewis lung carcinoma (LLC) cells form less bulky metastatic deposits in aged mice (>52 weeks) relative to their young (4–6 weeks) counterparts. Serial selection of LLC cells for increased metastatic capability in either young or old mice led in both cases to exaggerated growth of pulmonary nodules after only 5 cycles of in vivo passage. The respective metastatic cellular variants established in young (Y-series) or old (O-series) mice differed in cell morphology and constitutive activity of growth factor receptors, especially phospho-PDGFRa and phospho-EPHA7. [...

Cancer cells induced to express mesenchymal phenotype release exosome-like extracellular vesicles carrying tissue factor

Aggressive epithelial cancer cells frequently adopt mesenchymal characteristics and exhibit aberrant interactions with their surroundings, including the vasculature. Whether the release/uptake of extracellular vesicles (EVs) plays a role during these processes has not been studied. EVs are heterogeneous membrane structures that originate either at the surface (microparticles), or within (exosomes) activated or transformed cells, and are involved in intercellular trafficking of bioactive molecules. Here, we show that epithelial cancer cells (A431, DLD-1) adopt mesenchymal features (epithelial-to-mesenchymal transition-like state) upon activation of epidermal growth factor receptor (EGFR) coupled with blockade of E-cadherin. [...

Quantitative proteomics and biological activity of extracellular vesicles engineered to express SARS‐CoV‐2 spike protein

Abstract SARS‐CoV‐2 viral infection led to the devastating COVID‐19 pandemic, where illness stemmed from interactions between virions and recipient host cells resulting in multi‐layered pathological consequences. The role of the infection portal is now understood to be the cellular angiotensin converting enzyme‐2 (ACE2) receptor, which binds to viral spike (S) protein initiating virion internalisation process. Since SARS‐CoV‐2 virions bear some resemblance to endogenously produced small extracellular vesicles (sEVs) we reasoned that EVs engineered to express S protein (viral mimics) may interfere with viral infection. Here, we report generation of HEK293T cells producing sEVs enriched for transmembrane S‐protein tagged with green fluorescent protein (S/GFP). Strikingly, S protein drove the GFP tag to the membrane of sEVs, while GFP alone was not efficiently included in the sEV cargo. High‐throughput quantitative proteomics revealed that S/GFP sEVs contained over 1000 proteins including canonical components of the exosomal pathway such as ALIX, syntenin‐1, and tetraspanins (CD81, CD9), but depleted for calnexin and cytochrome c. We found that 84 sEV proteins were significantly altered by the presence of S/GFP. S protein expressing EVs efficiently adhered to target cells in an ACE2‐dependent manner, but they were poorly internalised. Importantly, prolonged administration of S/GFP EV to K18‐hACE2 mice provided a significant protection against SARS‐CoV‐2 infection. Thus, the generation of sEV containing S protein can be considered as a novel therapeutic approach in reducing the transmission of SARS‐CoV‐2

The Friedreich Ataxia Critical Region Spans A 150-kb Interval on Chromosome 9q13

By analysis of crossovers in key recombinant families and by homozygosity analysis of inbred families, the Friedreich ataxia (FRDA) locus was localized in a 300-kb interval between the X104 gene and the microsatellite marker FR8 (D9S888). By homology searches of the sequence databases, we identified X104 as the human tight junction protein ZO-2 gene. We generated a largescale physical map of the FRDA region by pulsed-field gel electrophoresis analysis of genomic DNA and of three YAC clones derived from different libraries, and we constructed an uninterrupted cosmid contig spanning the FRDA locus. The cAMP-dependent protein kinase γ-catalytic subunit gene was identified within the critical FRDA interval, but it was excluded as candidate because of its biological properties and because of lack of mutations in FRDA patients. Six new polymorphic markers were isolated between FR2 (D9S886) and FR8 (D9S888), which were used for homozygosity analysis in a family in which parents of an affected child are distantly related. An ancient recombination involving the centromeric FRDA flanking markers had been previously demonstrated in this family. Homozygosity analysis indicated that the FRDA gene is localized in the telomeric 150 kb of the FR2-FR8 interval