Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilian-Universität München Characterisation of apoptosis signal transduction induced by the marine compound cephalostatin 1 in leukemic cells Irina Melanie Müller aus Forchheim / Ofr. 2004 Erklärung Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom 29. Januar 1998 von Frau Prof. Dr. A. M. Vollmar betreut. Ehrenwörtliche Versicherung Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe angefertigt. München, den 04.03.2004 Irina Müller Dissertation eingereicht am: 04.03.2004 1. Gutachter: Frau Prof. Dr. A. M. Vollmar 2. Gutachter: Herr Prof. Dr. E. Wagner Mündliche Prüfung am: 15.04.2004 I. CONTENTS I. Contents ..................................................................................................... I II. Introduction ............................................................................................... 1 1. Background - Marine Compounds in Cancer Therapy ......................................1 2. The Family of Cephalostatins ..............................................................................3 2.1 Cephalodiscus gilchristi Ridewood................................................... 5 2.2 Biological activity of cephalostatin 1................................................. 7 3. Aim of the work .....................................................................................................8 4. Apoptosis / Programmed Cell Death and Necrosis ...........................................8 5. Signal transduction in Apoptosis......................................................................10 5.1 Caspases ....................................................................................... 11 5.2 Extrinsic Pathway / Death Receptor Pathway ................................ 13 5.3 Intrinsic Pathway / Mitochondrial Pathway ..................................... 15 5.3.1 The Bcl-2 Family ..................................................................... 17 5.3.2 Smac / DIABLO....................................................................... 19 5.4 ER-mediated Pathway.................................................................... 20 III. Materials and Methods........................................................................ 23 1. Materials and Solutions......................................................................................23 2. Cephalostatin 1 ...................................................................................................23 2.1 Isolation.......................................................................................... 23 3. Cell culture...........................................................................................................24 3.1 Cell Lines ....................................................................................... 24 3.2 Cell Culture Techniques ................................................................. 24 3.2.1 Freezing and thawing.............................................................. 24 3.2.2 Determination of cell concentration......................................... 25 3.2.3 Splitting and seeding............................................................... 25 4. Cytotoxicity Assays ............................................................................................26 4.1 MTT Assay ..................................................................................... 26 4.2 Propidium Iodide Exclusion Assay ................................................. 27 5. Quantification of Apoptosis by Flow Cytometry..............................................28 5.1 Introduction to Flow Cytometry....................................................... 28 5.2 Quantification by Staining with Propidium Iodide ........................... 29 5.3 Quantification by Staining with Annexin V ...................................... 31 5.4 Measurement of Mitochondrial Potential Dissipation...................... 32 II I Contents 6. Microscopy ..........................................................................................................33 6.1 Light Microscopy ............................................................................ 33 6.2 Fluorescence Microscopy............................................................... 33 6.2.1 Hoechst staining...................................................................... 33 6.2.2 JC1 staining ............................................................................ 34 6.3 Confocal Laser Scanning Microscopy (CLSM)............................... 34 6.4 Transmission Electron Microscopy................................................. 35 7. Caspase Activity Assay......................................................................................38 8. Western Blot Analysis ........................................................................................39 8.1 Preparation of Samples.................................................................. 39 8.1.1 Whole cell lysates ................................................................... 39 8.1.2 Cytosolic and particulate fractions........................................... 40 8.2 Protein Determination..................................................................... 41 8.3 SDS-PAGE..................................................................................... 42 8.4 Western Blotting and Detection of Proteins.................................... 44 8.5 Coomassie Staining of Gels ........................................................... 46 8.6 Membrane Stripping ....................................................................... 47 9. Immunoprecipitation...........................................................................................47 10. Comet Assay / Single Cell Gel Electrophoresis ...........................................48 11. Statistics ..........................................................................................................49 IV. Results ................................................................................................. 50 1. Cytotoxic Activity of Cephalostatin 1................................................................50 2. Apoptosis-Induction by Cephalostatin 1 ..........................................................50 2.1 Morphologic Alterations.................................................................. 51 2.1.1 Light microscopic analysis of cephalostatin 1-treated cells ..... 51 2.1.2 Morphologic analysis per flow cytometry................................. 51 2.1.3 Morphologic changes of the nucleus....................................... 52 2.1.4 Phosphatidylserine exposure .................................................. 53 2.2 Quantitative Analysis of Cephalostatin 1-Induced Apoptosis ......... 54 3. Signal Transduction in Cephalostatin 1-Induced Apoptosis ..........................56 3.1 Caspases are Essential for Cephalostatin 1-Induced Apoptosis ... 56 3.1.1 Inhibition of apoptosis by zVADfmk......................................... 57 3.2 Involvement of the Cd95 Pathway.................................................. 58 3.2.1 Activation of caspase-8 ........................................................... 58 3.2.2 Apoptosis induction in CD95 deficient cells............................. 59 3.3 Involvement of the Mitochondria .................................................... 61 3.3.1 Morphological alterations of cephalostatin 1-activated mitochondria.......................................................................................... 61 3.3.2 Alterations of the mitochondrial membrane potential .............. 63 3.3.3 Activation of caspase-9 ........................................................... 65 I Contents III 3.3.4 Release of apoptogenic factors from the intermembrane space . ................................................................................................ 65 3.3.5 Formation of an apoptosome .................................................. 68 3.3.6 Necessity of caspase-9 for cephalostatin 1-induces apoptosis 69 3.3.7 Overexpression of anti-apoptotic proteins Bcl-2 and Bcl-xL..... 70 3.3.8 Inactivation of Bcl-2................................................................. 74 3.4 Involvement of the Endoplasmic Reticulum.................................... 80 3.4.1 Cephalostatin 1 elicits ER stress............................................. 80 3.4.2 Activation of caspase-12 ......................................................... 81 V. Discussion............................................................................................... 83 Characterisation of the Cell Death Induced by Cephalostatin 1.............................83 Signal Transduction Pathways in Cephalostatin 1-Induced Apoptosis ................84 1.1 Involvement of CD95 Pathway ....................................................... 84 1.2 Involvement of Mitochondria in Cephalostatin 1-Induced Apoptosis .. ....................................................................................................... 85 1.3 Involvement of the Endoplasmic Reticulum.................................... 90 Regulation of Cephalostatin 1-Induced Apoptosis..................................................93 VI. Summary and Outlook ........................................................................ 96 VII. Appendix .............................................................................................. 98 1. Abbreviations ......................................................................................................98