Apoptosis: Architecture of chromosomAl territories in Apoptotic cells E. Bártová •Institute of Biophysics Academy of Sciences of the Czech Republic Cellular death-by-suicide is part of normal development, and is termed apoptosis or programmed cell death (PCD). Cysteine Aspartate Specific ProteASEs – caspases – are active in apoptosis, as are p53, a tumor suppressor gene, and FAS gene, which is member 6 of the tumor necrosis factor receptor superfamily (TNF). In contrast to apoptosis, necrosis is cell death that results from cytotoxic, injurious stresses that are too severe for correction by the cellular stress response. Apoptosis is a part of normal cell turnover and tissue homeostasis „History“ of molecular biology of cell death Horvitz (1992-3) identification of „cell death genes“ in Caenorhabditis elegans {ced-3 (ICE), ced-4 (0), ced-9 (bcl-2)} (Cerretti 1992, Thornberry 1992) uncovering of the homology between ced-3 gene product and ICE (interleukin-1b converting enzyme)] protease 1990 Discovery of new family of mammalian cysteine proteases - CASPASES Kerr et al., 1972: Identification of the cell death APOPTOSIS Kerr, Wylie and Currie Apoptosis: a basic biological pehenomenon with wide-ranging implications in tissue kinetics. Br. J.Cancer 1972;26:239-257 Apoptosis is involved in a wide range of physiological and pathological processes. > Development (embryonic, neuronal development) > Inflammation and involution of tissues > In the immune system (Apoptosis is employed as a method of cytotoxic T-cell mediated killing of infected cells) > In ageing Apoptosis plays a pivotal role in the pathophysiology of ageing'. The free radical theory of ageing links senescence to damage inflicted by superoxide-derived radicals and other oxidants generated primarily in mitochondrial respiration. The mitochondrial theory of ageing, proposes that ageing is the result of accumulated free radical damage to mitochondrial DNA (mtDNA). The accumulation of errors in mtDNA leads to errors in the polypeptides encoded by mtDNA, i.e., the four mitochondrial enzymatic complexes. Defective complexes produce more free radicals leading to a vicious cycle of increasing mtDNA damage, radical generation, and possibly apoptosis Apoptosis in contrast to necrotic cell death Roche: Cell Death - Apoptosis and Necrosis Uvolnění lysosomálních enzymů Nuclear morfology in HL-60 cells (P. Mlejnek 2001) I) II) III) I) Control II) Apoptosis III) Necrosis Cell death classification by Clarke CM – cyt. membrane J – nuclei M – mitochondrion ER – endopl. reticulum GA – Golgy complex L – lysosomes Apoptosis Autophagy Nelysosomal disintegration Cell death classification by Clarke  Apoptosis  Autophagy  Nonlysosomal disintegration - heterophagy, final cell destruction is done by lysosomes of other cells - final cell destruction is done by its own lysosomes - cell destruction is mediated by unknown nonlysosomal proteases Anoikis is a form of programmed cell death which is induced by anchorage-dependent cells detaching from the surrounding extracellular matrix (ECM)[1]. Usually cells stay close to the tissue to which they belong since the communication between proximal cells as well as between cells and ECM provide essential signals for growth or survival. When cells are detached from the ECM, i.e. there is a loss of normal cell-matrix interactions, they may undergo anoikis. However, metastatic tumor cells may escape from anoikis and invade other organs. Morphological features of apoptosis Scanning electron micrograph Transmission electron micrograph C-Knudson@uniowa.edu Apoptotic DNA degradation is followed by phogocytosis of apoptotic bodies Rich et al., Nature 2000 Hengartner M.O., Nature 2000 Two major apoptotic pathways in mammalian cells Death-receptor pathway: Death receptor superfamily: CD95 receptor and tumour necrosis factor receptor. CD95 ligand binds to CD95 receptor to form death inducing signaling complex. This complex recruits via the adaptor molecule FADD (Fas-associated death domain protein). Procaspase 8 binds to this complex in order to activate Caspase-8 and subsequenty activation of Caspase-3 is induced. Activation of procaspase-8 can be blocked through degenerate caspase homoloque c-FLIP. Hengartner M.O., Nature 2000 Two major apoptotic pathways in mammalian cells The mitochondrial pathway - activated after DNA damage - proapoptotic members of Bcl-2 family, located on the surface of mitochondria, are activated - Cytochrome c is released from mitochondria and forms complex with Apaf-1 and Procaspase 9. - The complex is called APOPTOSOME. Both apoptotic pathways converge on the level of Caspase-3 activation Caspase-3 activation is antagonized by IAP released from mitochondria Sigma (Apoptosis and Life Science) www.cellsignal.com DNA repair Poly(ADP-ribosyl)ation and apoptosis Anti-PARP p85 fragment pAb DNA fragmentation during apoptosis 1. High molecular weight DNA fragmentation (50-300 kbp) 2. Oligonucleosomal DNA fragmentation (180-200 bp) 3. Single- strand cleavage Bortner C.D. et al., 1995 APOPTOSIS DETECTION DNA fragmentation test Large and oligonucleosomal DNA fragmentation in apoptotic cells (M. Fojtová, BFÚ Brno) Field inversion electrophoresis (FIGE) DNA fragmentation test 1. Etoposide 2. Cis-platin 3. Vincristine 4. Gamma-irradiation 5. Serum deprivation Apoptosis was detected in human erythroleukemia cell line K-562 and human retinoblastoma cell line Y79 Control Serum deprivation Etoposide Annexin V / PI Hoechst33342 / PI ControlEtoposide Anexin V binds to phosphatidylserines that are traslocated from the inner side od the plasma membrane to the cell surface soon after the induction of apoptosis CaspSCREEN (tm) BioVision kit TMRE Etoposide Control The results of TUNEL test Nuclear organisation of chromosomal territories (Cremer T. and Cremer C., 2001) Territory of chromosome 11 and 17 Territory of chromosome 3 Arcitecture of chromosomal territories during apoptosis Retinoblastoma Y79 cells and HSR MYCN (2p24) TUNEL and PI staining of fixed cells Apoptosis in patient suffering from retinoblastoma TUNEL and DAPI staining A. Liepins/SPL Conclusions * Differences in DNA fragmentation * Differences in the number of nuclear apoptotic bodies * Chromosomal territories cleaved into high molecular DNA fragments were variably disassembled into apoptotic bodies whose induction is the main effort of anticancer therapy. * Apoptotic nuclear segmentation can be observed at centromeric regions. * Disassembly of chromosomal territories was also found in pre-apoptotic (TUNEL positive) nuclei. * Apoptosis can be observed not only after experimental and/or clinical treatment but also spontaneously.