Dissertation der Fakultät für Biologie der Ludwig‐Maximilians‐Universität München Functional characterization of B-cell receptor associated protein 31 in cancer cells Olga Nagło (geb. Chojnacka) München, 2019 1 Diese Dissertation wurde angefertigt unter der Leitung von Prof. Dr. Angelika Vollmar im Bereich der Pharmazeutischen Biologie an der Ludwig‐Maximilians‐Universität München Erstgutachterin: Prof. Dr. Barbara Conradt Zweitgutachterin: Prof. Dr. Angelika Vollmar Tag der Abgabe: 06.06.2019 Tag der mündlichen Prüfung: 13.08.2019 ERKLÄRUNG Ich versichere hiermit an Eides statt, dass meine Dissertation selbständig und ohne unerlaubte Hilfsmittel angefertigt worden ist. Die vorliegende Dissertation wurde weder ganz, noch teilweise bei einer anderen Prüfungskommission vorgelegt. Ich habe noch zu keinem früheren Zeitpunkt versucht, eine Dissertation einzureichen oder an einer Doktorprüfung teilzunehmen. München, den 06.06.2019 __________________________ Olga Nagło 2 1. TABLE OF CONTENTS 1. TABLE OF CONTENTS ................................................................................................... 3 2. INTRODUCTION ............................................................................................................. 7 2.1 CANCER INCIDENCE AND MORTALITY IN 2018 ............................................................ 7 2.2 HALLMARKS OF CANCER ............................................................................................. 7 2.3 RESISTANCE TO THE COMMON CHEMOTHERAPEUTICS AS A SERIOUS ISSUE ................. 8 2.4 RE-SENSITIZATION OF CANCEROUS CELLS TOWARDS TREATMENT THROUGH INDUCTION OF ER-STRESS ..................................................................................................... 10 2.5 SMALL MOLECULES – T8 AND PS89 .......................................................................... 10 2.6 FUNCTION OF B CELL RECEPTOR - ASSOCIATED PROTEIN (BAP31) PROTEIN ............. 12 3. MATERIALS AND METHODS ..................................................................................... 15 3.1 MATERIALS ............................................................................................................... 15 3.1.1 Cells ..................................................................................................................... 15 3.1.2 Compounds .......................................................................................................... 15 3.1.3 Chemicals and Reagents ...................................................................................... 16 3.1.4 Primary antibodies ............................................................................................... 20 3.1.5 Secondary antibodies ........................................................................................... 21 3.1.6 Technical equipment ............................................................................................ 22 3.1.7 Software ............................................................................................................... 23 3.2 CELL CULTURE .......................................................................................................... 24 3.2.1 Maintenance of cell lines ..................................................................................... 24 3.2.2 Freezing and thawing process .............................................................................. 25 3.2.3 Isolation of Peripheral Blood Mononuclear Cells (PBMCs) ............................... 25 3.2.4 Generation and isolation of patient derived xenograft cells (PDX) ..................... 26 3.2.5 Stimulation with compounds ............................................................................... 26 3.3 PROLIFERATION AND ATTACHMENT ASSAYS ............................................................. 26 3.3.1 Cell-Titer Blue® viability assay .......................................................................... 26 3.3.2 ViCell – counting of the cell number ................................................................... 27 3.3.3 xCELLigence – the real-time analysis of the cellular growth and attachment .... 27 3 3.3.4 Attachment analysis – crystal violet staining ....................................................... 27 3.3.5 Colony formation assay ....................................................................................... 27 3.4 IMMUNOBLOTTING .................................................................................................... 28 3.4.1 Preparation of samples ......................................................................................... 28 3.4.2 Preparation of samples for the cytosol/mitochondrial fractionation .................... 28 3.4.3 Preparation of samples for the cytosol/nuclear fractionation. ............................. 29 3.4.4 Determination of protein concentration ............................................................... 30 3.4.5 Sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) ..... 31 3.4.6 Blotting process ................................................................................................... 32 3.4.7 Protein detection .................................................................................................. 33 3.5 FLOW CYTOMETRY .................................................................................................... 33 3.5.1 Analysis of apoptosis ........................................................................................... 33 3.5.2 Measurement of cytosolic calcium level .............................................................. 34 3.5.3 Measurement of Reactive Oxygen Species (ROS) level ..................................... 35 3.5.4 Identification of apoptosis in the CD34+ cells .................................................... 36 3.6 TRANSIENT TRANSFECTION OF CELLS ........................................................................ 36 3.6.1 Transfection with siRNA ..................................................................................... 36 3.6.2 Transfection with plasmid.................................................................................... 37 3.7 GENOME EDITING USING THE CRISPR-CAS9 TECHNIQUE ......................................... 37 3.7.1 Design of targeting components. ......................................................................... 37 3.7.2 Cloning of oligos into Cas9 plasmid .................................................................... 39 3.7.3 Transformation of plasmids into E. Coli.............................................................. 41 3.7.4 Determining Genome Targeting Efficiency using T7 Endonuclease I ................ 41 3.7.5 Transfection and selection of clones. ................................................................... 42 3.7.6 2.8. The polymerase chain reaction (PCR) .......................................................... 42 3.8 MIGRATION OF CANCER CELLS .................................................................................. 43 3.8.1 The Boyden Chambers ......................................................................................... 43 3.8.2 Wound healing assay ........................................................................................... 43 3.9 LUCIFERASE DOUBLE REPORTER GENE ASSAY - SPLICING ....................................... 43 3.10 IMMUNOSTAINING AND CONFOCAL MICROSCOPY ...................................................... 44 3.11 RECYCLING OF THE TRANSFERRIN RECEPTOR IN CANCER CELLS. .............................. 44 3.12 FORMATION OF LIPOSOMES CONTAINING PHOSPHATIDYLINOSITOL (PI) MIX ............. 45 3.13 RESCUE OF THE SORAPHEN A TREATMENT EFFECT ................................................... 45 4 3.14 IDENTIFICATION OF BAP31 INTERACTION PARTNERS APPLYING YEAST 2 HYBRID APPROACH ............................................................................................................................. 46 4. AIMS OF THE STUDY .................................................................................................. 47 5. RESULTS ........................................................................................................................ 48 5.1 INVOLVEMENT OF BAP31 IN THE CHEMO-SENSITIZATION OF VARIOUS LEUKEMIA TYPES TOWARDS COMMONLY USED CHEMOTHERAPEUTICS ................................................... 48 5.1.1 Combination of various cytostatics with PS89 enhances their anti-cancer properties in different leukemia types but not in hematopoietic cells. ............................ 48 5.1.2 Caspase-8 and BAP31 activation constitutes a platform for the induction of apoptosis in leukemia cells upon the treatment with the combination of PS89 and cytostatic. ......................................................................................................................... 52 5.1.3 The pro-apoptotic cross-talk between Endoplasmic Reticulum (ER) and mitochondria. ................................................................................................................... 54 5.1.4 The cytochrome c release induces an activation of the death protease. ............... 57 5.1.5 The summary of the effect of BAP31 activation on the chemo-sensitization of leukemic cells towards cytostatic treatment. ................................................................... 58 5.2 THE FUNCTION OF BAP31 IN HEPATOCELLULAR CARCINOMA................................... 59 5.2.1 Generation of the BAP31 deficient cell line. ......................................................