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Molecular Mechanism of Pe5-Induced Cytotoxicity and Generation of New Cytotoxic Nuclear-Directed

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Molecular Mechanism of Pe5-Induced Cytotoxicity and Generation of New Cytotoxic Nuclear-Directed MOLECULAR MECHANISM OF PE5-INDUCED CYTOTOXICITY AND GENERATION OF NEW CYTOTOXIC NUCLEAR-DIRECTED Anna Vert Company Dipòsit legal: Gi. 1801-2014 http://hdl.handle.net/10803/283575 ADVERTIMENT. L'accés als continguts d'aquesta tesi doctoral i la seva utilització ha de respectar els drets de la persona autora. Pot ser utilitzada per a consulta o estudi personal, així com en activitats o materials d'investigació i docència en els termes establerts a l'art. 32 del Text Refós de la Llei de Propietat Intel·lectual (RDL 1/1996). Per altres utilitzacions es requereix l'autorització prèvia i expressa de la persona autora. En qualsevol cas, en la utilització dels seus continguts caldrà indicar de forma clara el nom i cognoms de la persona autora i el títol de la tesi doctoral. No s'autoritza la seva reproducció o altres formes d'explotació efectuades amb finalitats de lucre ni la seva comunicació pública des d'un lloc aliè al servei TDX. Tampoc s'autoritza la presentació del seu contingut en una finestra o marc aliè a TDX (framing). Aquesta reserva de drets afecta tant als continguts de la tesi com als seus resums i índexs. ADVERTENCIA. El acceso a los contenidos de esta tesis doctoral y su utilización debe respetar los derechos de la persona autora. Puede ser utilizada para consulta o estudio personal, así como en actividades o materiales de investigación y docencia en los términos establecidos en el art. 32 del Texto Refundido de la Ley de Propiedad Intelectual (RDL 1/1996). Para otros usos se requiere la autorización previa y expresa de la persona autora. En cualquier caso, en la utilización de sus contenidos se deberá indicar de forma clara el nombre y apellidos de la persona autora y el título de la tesis doctoral. No se autoriza su reproducción u otras formas de explotación efectuadas con fines lucrativos ni su comunicación pública desde un sitio ajeno al servicio TDR. Tampoco se autoriza la presentación de su contenido en una ventana o marco ajeno a TDR (framing). Esta reserva de derechos afecta tanto al contenido de la tesis como a sus resúmenes e índices. WARNING. Access to the contents of this doctoral thesis and its use must respect the rights of the author. It can be used for reference or private study, as well as research and learning activities or materials in the terms established by the 32nd article of the Spanish Consolidated Copyright Act (RDL 1/1996). Express and previous authorization of the author is required for any other uses. In any case, when using its content, full name of the author and title of the thesis must be clearly indicated. Reproduction or other forms of for profit use or public communication from outside TDX service is not allowed. Presentation of its content in a window or frame external to TDX (framing) is not authorized either. These rights affect both the content of the thesis and its abstracts and indexes. PhD Thesis Molecular mechanism of PE5-induced cytotoxicity and generation of new cytotoxic nuclear-directed ribonuclease variants Anna Vert Company - 2014 Ciències Experimentals i Sostenibilitat Dirigida per Dirigida per Dra. Maria Vilanova Brugués Dr. Antoni Benito Mundet Catedràtica de Bioquímica Professor Titular de Bioquímica i Biologia Molecular i Biologia Molecular Memòria presentada per optar al títol de doctora per la Universitat de Girona Aquesta tesi ha estat realitzada amb el suport del Ministerio de Educación y Ciencia (beca AP2007-01944 i projecte BFU2006-15543-CO2-02/BMC) i del Ministerio de Ciencia e Innovación (projecte BFU2009-06935/BMC). Moltes gràcies a tots els que m’heu acompanyat. Contents List of tables .................................................................................................................. 11 List of figures ................................................................................................................ 13 List of abbreviations .................................................................................................... 15 Publication arising from this thesis ............................................................................ 18 Summary ........................................................................................................................ 19 Resum ............................................................................................................................. 21 Resumen ........................................................................................................................ 23 Introduction ........................................................................................... 25 RNases as potential antitumor drugs ......................................................................... 28 Natural antitumor RNases .......................................................................................... 29 Onconase ................................................................................................................. 29 Bovine seminal RNase ........................................................................................... 34 Amphinase ............................................................................................................... 35 jSBL and cSBL lectins ............................................................................................ 35 Engineered cytotoxic RNases ..................................................................................... 36 PE5 variant .............................................................................................................. 37 Gene and miRNA expression profiles of cells treated with RNases .................... 39 Objectives ............................................................................................... 41 Methods .................................................................................................. 45 Construction of RNase variants ................................................................................. 47 RNase expression and purification ............................................................................ 50 Determination of thermal stability ............................................................................. 51 Determination of steady-state kinetic parameters ................................................... 52 RNase inhibitor binding assay .................................................................................... 52 Cell lines and culture conditions ................................................................................ 52 Cell proliferation assays ............................................................................................... 53 Cytosolic and nuclear RNA degradation assay ......................................................... 54 Phosphatidylserine exposure assay ............................................................................ 54 09 Contents Procaspase activation assay .......................................................................................... 55 Western blot analysis .................................................................................................... 55 Doxorubicin accumulation assay ................................................................................ 56 RNA isolation for microarray analysis ....................................................................... 57 Gene expression microarrays ...................................................................................... 57 miRNA microarrays ...................................................................................................... 59 Results .................................................................................................. 61 Study of the molecular mechanism of PE5- and onconase- induced cytotoxicity .. 63 Selection of treatment conditions ......................................................................... 63 Generation of gene expression profiles ............................................................... 66 Identification of PE5 and onconase differentially expressed genes ................. 71 Gene ontology analysis and KEGG pathway annotation of PE5 and onconase differentially expressed genes ............................................................... 78 Generation of miRNA expression profiles .......................................................... 84 Identification of PE5 and onconase differentially expressed miRNAs ........... 88 Generation of new cytotoxic HP-RNase variants directed to the nucleus ........... 91 Half of the residues mutated in PE5 can be back-mutated to HP-RNase without decreasing its cytotoxicity ........................................................................ 91 The introduction of an additional NLS may increase the cytotoxicity of PE5 depending on the insertion site ................................................................ 93 The cytotoxicity of NLSPE5 and PE10 is dependent on the cleavage of nuclear RNA ........................................................................................................ 96 The effects induced by NLSPE5 and PE10 on cancer cells are analogous to those of PE5 ........................................................................................................ 98 Discussion .......................................................................................... 103 Conclusions ........................................................................................ 145 References .............................................................................................
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