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Lez Tumori1b Cellbiolwnt.Pptx 06/04/19 Come studio la migrazione? INVASION ASSAY Boyden Chamber Transwell Assay Count Cells under microscope and/or quantify after extraction Solution colorimetric (560nm) or Fluorescent (480nm) 1 06/04/19 Boyden Chamber Assay CytochalasinD Micotoxin that Inhibits actin polimerization Figure 1. Human Fibrosarcoma HT-1080 Cell Invasion. HT-1080 and NIH3T3 (negave control) were seeded at 300,000 cells/ well and allowed to invade toward 10% FBS for 24 hrs in the presence or absence of 2 μM Cytochalasin D. Invasive cells on the boRom of the invasion membrane were stained (top panel picture) and quanCfied at OD 560nm aer eXtracCon (boRom panel figure). 2 06/04/19 Cell Migration CellProfiler cell Image analysis software 3 06/04/19 FibroblasC embrionali di topo hp://www.lgcpromochem.com/atcc/ ATCC American Type Culture Collection is a nonprofit organization which collects, stores, and distributes standard reference microorganisms, cell lines and other materials for research and development 4 06/04/19 Un focus di fibroblasti embrionali di pollo indotto dal virus del sarcoma di Rous Focus Assay per perdita di inibizione da contao Test di Trasformazione cellulare Test di formazione di foci (Harry Rubin e Howard Temin, 1958): foci di cellule addensate e morfologicamente alterate. Test basato sulla perdita di inibizione da contatto su fibroblasti embrionali di topo BALB/c 3T3 Crescita in agar: Le cellule trasformate non debbono attaccarsi ad Una superficie solida prima di potersi dividere. Test basato sulla Indipendenza da legame integrine-matrice extracellulare Ridotta richiesta di siero: richiedono meno del 5% di siero. Basato su attivazione di segnali di proliferazione e capacità autocrine 5 06/04/19 Crescita senza supporto Mammosfere : tumor stem cells 6 06/04/19 Mammospheres 1st generation MCF7 p9 Ctrl Metformin 10 mM Inhibition of 100 micron Notch???? diameter These ‘tumor stem cells’ share the properties of self-renewal Only a minority of cells in human cancers and differentiation with their normal are capable of self renewal. stem cell counterparts, although in tumors these processes are deregulated. 100 cells Epitelial, Myoep., Alveolar Differenziazione cellulare Differenziazione struRurale 7 06/04/19 CADERINE Test di Trasformazione cellulare Test di formazione di foci (Harry Rubin e Howard Temin, 1958): foci di cellule addensate e morfologicamente alterate. Test basato sulla perdita di inibizione da contatto su fibroblasti BALB/c 3T3 Crescita in agar: Le cellule trasformate non hanno bisogno di attaccarsi ad una superficie solida prima di potersi dividere. Test basato sulla Indipendenza da legame integrine-matrice extracellulare Ridotta richiesta di siero: richiedono meno del 5% di siero. Basato su attivazione di segnali di proliferazione (RAS) e capacità autocrine 8 06/04/19 Omofiliche -Calcio I 4 Cpi di Molecole di carboidra Adesione Cell-cell Eterofiliche -Calcio Molecole adesione cell-cell AJ Caderina-Actina DS Cad-filamenti intermedi Integrine- filamenti intermedi 9 06/04/19 The integrity of the CCC cadherin-catenin complex is negatively regulated by phosphorylation of beta-catenin by receptor tyrosine kinases (RTKs) and cytoplasmic tyrosine kinases (Fer, Fyn, Yes, and Src), which leads to dissociation of the cadherin-catenin complex Recettori per fattori di crescita: giunzioni serrate (tight junction) TGFβRI e II, IGF-1R, EGFR, c-Met Claudina, occludina F-actina giunzioni aderenti (adherens junctions) p120 cat. E-caderina β cat. vinculina α cat. F-actina 10 06/04/19 Importanza delle caderine Nei tumori diminuiscono per varie cause: Mutazioni LOF Overespressione di fattori che le destabilizzano Overespressione di repressori trascrizionali Soppressione epigenetica della espressione Degradazione da proteasi 11 06/04/19 CADH1=E cadherin chr.16q22 30 mutazioni germinali in tumori, OMIM 192090 UniProtKB: P12830 882AA : 25/30 mutazioni inavanC- 5/30 sono missense La E-caderina è un TSG: Le mutazioni sono distribuite lungo tutto il gene Le mutazioni sono missense, non sense, splicing, frame shift (LOF) 60% dei tumori presenta mutazioni in CDH1, insieme con concomitante perdita dell’allele wt (LOH). Spesso si riscontra perdita delle caderine per attivazione di proteasi da influsso di calcio (intracellulare), caspase3 o gamma-secretase. Oppure metallo proteasi di matrice 12 06/04/19 Mol Cell Biol. 2007 May;27(10):3804-16. Epub 2007 Mar 12.Casein kinase 1 is a novel negative regulator of E-cadherin-based cell-cell contacts.Dupre-Crochet S, Figueroa A, Hogan C, Ferber EC, Bialucha CU, Adams J, Richardson EC, Fujita Y. E-CADERINA P12830 S846D CK1 casein kinase IC261 Fosforila S846 Phosphorylated S846 E-CADH has weaker interaction with beta-catenin MYC, CyclinD Le caderine prevengono la localizzazione LRP Low Density nucleare della Lipoprotein Beta-catenina receptor Giunzioni AderenC WNT: 19 human Orthologs 13 06/04/19 A road to cancer : 5ghtly regulated self renewal system subverted in cancer Wingless in Drosophila and its ORTHOLOG int-1 in mouse (InserCon of Mammary Tumor Virus) WNT1 protooncogene - 19 ortholog genes in homo sapiens- secreted lipid-modified signaling glycoproteins (350–400 aa) WNT are a family of CYSTEIN RICH, secreted glycoproteins. Palmitoylation in Serine is required for efficient binding to frizzled receptors. Palmitoylation is necessary for proper trafficking to cell surface. "Fay acid modificaon of Wnt1 and Wnt3a at serine is prerequisite for lipidaon at cysteine and is essenCal for Wnt signalling." Doubravska L., Krausova M., Gradl D., Vojtechova M., Tumova L., Lukas J., Valenta T., Pospichalova V., Fafilek B., Plachy J., Sebesta O., Korinek V. Cell. Signal. 23:837-848(2011) WNT1 mutaons in early-onset osteoporosis and osteogenesis imperfecta. Laine C.M., Joeng K.S., Campeau P.M., Kiviranta R., Tarkkonen K., Grover M., Lu J.T., Pekkinen M., Wessman M., Heino T.J., Nieminen- Pihala V., Aronen M., Laine T., Kroeger H., Cole W.G., Lehesjoki A.E., Nevarez L., Krakow D., Curry C.J., Cohn D.H., Gibbs R.A., Lee B.H., MaekiCe O. This report idenCfies human skeletal diseases associated with mutaons in WNT1. In 10 family members with dominantly inherited, early-onset osteoporosis, we idenCfied a heterozygous missense mutaon in WNT1, c.652T→G (p.Cys218Gly). In a separate family with 2 siblings affected by recessive osteogenesis imperfecta, we idenCfied a homozygous nonsense muta:on, c.884C→A, p.Ser295*. In vitro, aberrant forms of the WNT1 protein showed impaired capacity to induce canonical WNT signaling, their target genes, and mineralizaon. In mice, Wnt1 was clearly eXpressed in bone marrow, especially in B-cell lineage and hematopoieCc progenitors; lineage tracing idenCfied the eXpression of the gene in a subset of osteocytes, suggesCng the presence of altered cross-talk in WNT signaling between the hematopoieCc and osteoblasCc lineage cells in these diseases. N. Engl. J. Med. 368:1809-1816(2013) C218G (i.e., NO LIPIDATION) : osteoporosis, LOF S224 O-palmitoyl serine N-linked (GlcNAc...) asparagine 29, 316, 346, 359 (WNT1 human P04628) 14 06/04/19 Three common lipid modifications that occur in the cytoplasm are N-myristoylation, S-palmitoylation, and prenylation S-palmitoylation is the covalent addition of a long-chain fatty acid to a cysteine residue via a thioester linkage Less frequent pamitoylation of Serine or threonine N-myristoylation is the covalent addition of the fatty acid myristate to an N-terminal glycine residue via an amide linkage Prenylation is the addition of an isoprenoid, either a C15 farnesyl or a C20 geranylgeranyl group, to a C-terminal cysteine residueor near the C-terminus via a thioether linkage. These lipid groups mediate protein attachment to membranes and their addition is specified by the amino acid sequence motifs CAAX, CC or CAC at the C-terminus. Prenylated proteins are estimated to represent 0.5% of all intracellular proteins. Prenylation of a cysteine in the CAAX context often leads to both proteolytic cleavage of the C-A bond and to the methylation of the new C-terminal cysteine residue. Both the cleaved propeptide and the accompanying methylation of the new C-terminal cysteine are annotated. https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1742-4658.2007.06056.x 15 06/04/19 P04628 (WNT1_HUMAN) the PROTO-ONCOGENE C218G (i.e., NO LIPIDATION) : osteoporosis, LOF The discovery of Wnt signaling was influenced by research on oncogenic retroviruses. In 1982, Roel Nusse and Harold Varmus infected mice with mouse mammary tumor virus MMTV in order to mutate mouse genes to see which mutated genes could cause breast tumors. They identified a new mouse proto- oncogene that they named int1 (integration 1). 16 06/04/19 Nusse R, van Ooyen A, CoX D, Fung YK, Varmus H (1984). "Mode of proviral acCvaon of a putave mammary oncogene (int-1) on mouse chromosome 15". Nature 307 (5947): 131–6. doi:10.1038/307131a0. PMID 6318122. A GOF mutaon Nüsslein-Volhard C, Wieschaus E (October 1980). "Mutaons affecCng segment number and polarity in Drosophila". Nature 287 (5785): 795–801. doi:10.1038/287795a0. PMID 6776413 A LOF mutant-Null mutaon at the wg locus 1995 Nobel Price Physiology and Medicine Eric Wieschaus Christiane Nüsslein-Volhard Eric Wieschaus at the European Molecular Biology Laboratory in Heidelberg , where he did his Nobel Prize-winning work. 17 06/04/19 Wnts are a conserved gene family found throughout the animal kingdom. That have been shown to be required during growth, tissue patterning, and homeostasis. In addition, inappropriate activation of Wnt-dependent gene expression in mammals can lead to numerous
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