Apoptosis in Development & Diseases PDF

Cell Death & Apoptosis Mol & Cell Biology MD105 Prof A. Stephanou  OBJECTIVES: The understand the types of cell death pathways How to assess cell death. To understand the physiological process associated with apoptosis How defects in the apoptotic pathways are asso...

Cell Death & Apoptosis Mol & Cell Biology MD105 Prof A. Stephanou  OBJECTIVES: The understand the types of cell death pathways How to assess cell death. To understand the physiological process associated with apoptosis How defects in the apoptotic pathways are associated with disease's  Cell Death The body is very good at maintaining a constant number of cells. So there has to exist mechanisms for ensuring other cells in the body are removed, when appropriate. Two major forms – Apoptosis - suicide - programmed cell death – Necrosis - killing - decay and destruction  18_18_sculpts_digits.jpg Cell Death -occurs more often than one imagines! a) Most embryo development involves programmed cell death.  18_19_tadpole_frog.jpg b) The tail of the tadpole is absorbed via apoptosis. Also, in adult multicellular organisms cell death is a regular occurrence. In humans EACH HOUR we lose many BILLIONS of cells via apoptosis. Most of these are healthy cells which have no defects. WHY? Development and regulation controls. i.e. B and T cells are removed that do not pass certain tests.  Fruit fly Worm Apoptosis and Diseases  Apoptotic changes Morphological changes in apoptosis Biochemical Changes in Apoptosis  18_20_Apoptosis_.jpg Apoptosis results in a quick and clean cell death, without damaging its neighbours, or eliciting an immune response. Every cell is equipped with the ‘cell death pathway’. Apoptosis is an intracellular proteolytic pathway. The DNA is broken into small 200 bp units. The cytoplasm shrinks. The mitochondria release cytochrome c. The outer surface of the plasma membrane gets coated with a different Death by Injury vs. Death by Suicide (Necrosis vs. Apoptosis) Macrophages- phagocytes  What is Apoptosis ? Programmed cell death（PCD) Necrosis is dna is cleaved randomly Apoptosis vs Necrosis ⚫ Cellular condensation Cellular swelling ⚫ Membranes remain intact Membranes are broken ⚫ Requires ATP ATP is depleted ⚫ Cell is phagocytosed, no Cell lyses, eliciting an tissue reaction inflammatory reaction ⚫ Ladder-like DNA DNA fragmentation is fragmentation random, or smeared ⚫ In vivo, individual cells In vivo, whole areas of appear affected the tissue are affected Morphological differences in apoptosis and necrosis  Necrosis: consequences of irreversible cell injury  Necrosis: consequences of irreversible cell injury  Necrosis Trauma (toxic chemicals, mechanical injury, heat, hypoxia) Loss of ability to regulate internal environment Ca2+ influx accompanied by swelling Alteration of protein activity Production of toxic compounds (activation of cyclooxygenases) arachadonic acid prostaglandins eicosanoids Inflammation What makes a cell commit suicide? withdrawal of positive signals (growth factors, Il-2) receipt of negative signals (increased levels of oxidants, DNA damage via X-ray or UV light, chemotherapeutic drugs, accumulation of improperly folded proteins, death activators such as: TNF-a, TNF-b, Fas/FasL) Steps in apoptosis: the decision to activate the pathway; the actual "suicide" of the cell; engulfment of the cell remains by specialized immune cells called phagocytes; degradation of engulfed cell. The actual steps in cell death require: condensing of the cell nucleus and breaking it into pieces condensing and fragmenting of cytoplasm into membrane bound apoptotic bodies; breaking chromosomes into fragments containing multiple number of nucleosomes (a nucleosome ladder) Apoptosis Triggered via Two Pathways Intrinsic or mitochondrial pathway Extrinsic or death receptor pathway Apoptotic Signalling  APOPTOSIS: Morphology organelle reduction membrane blebbing & changes mitochondrial cell leakage shrinkage nuclear chromatin fragmentation condensation Hacker., 2000  Biochemical Changes in Apoptosis ⚫ Caspase activation Have to be cleaved to be activated Ex western blotting which is antibodies ⚫ Endonuclease activation  Caspases Enzymatic proteins (proteases) which degrade other proteins are employed by apoptosis - caspases Made as inactive precursors - procaspases These are activated by other proteins when the right signal is received One caspase cleaves the lamin proteins resulting in the irreversible breakdown of the nuclear membrane. Caspases: (cysteine-containing aspartate-specific proteases) Most apoptotic proteolytic cleavage results from the action of caspases Caspases are activated by proteolytic cleavage Removal of prodomain and linker region Assembly of the large and small subunits into an active enzyme complex Two heterodimers interacting via the small subunits to form a tetramer with two catalytic sites Family members>14 Caspase functions and structure Apoptotic: Pathways Extrinsic Pathway - Death receptor mediated Apoptotic: Pathways Intrinsic Pathway – Mitochondrial mediated Ligand/death-induced cell death Ligand Receptor FasL Fas (CD95) TNF TNF-R TRAIL DR4 (Trail-R)  Ligand-induced cell death FasL “The death receptors” Ligand-induced trimerization Trail TNF Death Domains Death Effectors Induced proximity of Caspase 8 Activation of Caspase 8  The mitochondrial pathway Extrinsic linking Intrinsic DNA Fas Growth factor damage receptors Casp8 PI3K p53 Bid Akt casp3 Bid BAD Bax Make sure cyt c remains in the mitochondria , it’s a anti Bid casp9 IAPs Bcl2 Apaf1 ATP Bax Cyt.C casp3 Smac/ DIABLO H2O2 AIF Pollack etal., 2001  Assessing and Imaging Apoptosis Nuclear Apoptosis Assays by Flow Cytometry or microscopy Caspase Assays for Flow Cytometry or Western blotting Detection of apoptotic changes in DNA: Nucleic acid staining – nuclear morphology Detection of nuclear DNA fragmentation TUNEL staining (terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling) Molecular Probes, Inc. Single-cell electrophoresis (Comet assay)  Importance of Apoptosis Important in normal physiology / development – Development: Immune systems maturation, Morphogenesis, Neural development – Adult: Immune privilege, DNA Damage and wound repair. Excess apoptosis – Neurodegenerative diseases – Cardiac infarction Ex, alzimers Deficient apoptosis – Cancer – Autoimmunity  Other Forms of cell Death Autophagic Cell death Autophagy (self-eating) is observed in healthy and dying cells，whether autophagy plays a protective or a destructive role during an stress response? Necroptosis Necroptosis is a programmed form of necrotic cell death. Necrotic cell death has been considered a form of passive cell death. However, the discovery that TNFalpha mediated necrosis can be inhibited by a specific inhibitor of RIP1 kinase, necrostatin-1, led to the concept of necroptosis;.Necroptosis has now been established as a regulated necrotic cell death pathway controlled by RIP1 and RIP3 kinases  Autophagic cell death: instances of cell death that are accompanied by a massive cytoplasmic vacuolization In response to stress and during development, eukaryotic cells often activate autophagy, a mechanism whereby organelles and portion of the cytoplasm are sequestered in double-membraned vesicles (autophagosomes) that are delivered to lysosomes for degradation. Stress-induced autophagy most often exerts cytoprotective functions and favors the re-establishment of homeostasis and survival (a). In this setting, pharmacological or genetic inhibition of autophagy accelerates cell death. On the contrary, these interventions frequently inhibit developmental cell death, indicating that autophagy also constitutes a lethal mechanism that mediates ‘autophagic cell death’ (b)  Necroptosis plays a role in various pathological forms of cell death, including ischemic brain injury, neurodegenerative diseases and viral infections. Necroptosis leads to rapid plasma membrane permeabilization and to the release of cell contents and exposure of damage-associated molecular patterns (DAMPs). Apoptosis Necrosis Necroptosis Autophagy Autosis Ferroptosis Immunogenic cell death Lysozomal cell death \ Mitotic cell death Pyroptosis  Apoptosis excess Ischemia reperfusion injury Distinct initiator caspases are required for the induction of apoptosis in cardiac myocytes during ischaemia versus reperfusion injury. Stephanou A, Brar B, Liao Z, Scarabelli T, Knight RA, Latchman DS. Cell Death Differ. 2001 Apr;8(4):434-5. Apoptosis of endothelial cells precedes myocyte cell apoptosis in ischemia/reperfusion injury. Scarabelli T, Stephanou A, Rayment N, Pasini E, Comini L, Curello S, Ferrari R, Knight R, Latchman D. Circulation. 2001 Jul 17;104(3):253-6. Triggered by a atrioscarotic clot triggers heart attack Apoptosis excess following I/R First stage of a heart attack Triggers the exintric pathway Ischaemia Reperfusion  Cancer and Apoptosis (reduced) Evasion of apoptosis can lead to cancer Activate apoptosis in cancer cells, problem solved What is the molecular basis of cancer? Cancer is a genetic disease. Mutations in genes result in altered proteins during cell division Most cancers result from mutations in somatic and germline cells GENES PLAYING ROLE IN CANCER DEVELOPMENT Oncogenes P53 is a ex of a tumour sup res or gene Tumor suppressor genes DNA repair genes  Functions of Cellular Proto-Oncogenes 1. Secreted Growth Factors 2. Growth Factor Receptors  Oncogenes proto-oncogene = ras Oncogene = mutated ras Always activated Always stimulating proliferation  Tumor suppressor genes ⚫ Normal function - inhibit cell proliferation ⚫ Absence/inactivation of inhibitor --> cancer  TUMOR SUPPRESSOR GENES Disorders in which gene is affected Gene (locus) Function Familial Sporadic DCC (18q) cell surface unknown colorectal interactions cancer WT1 (11p) transcription Wilm’s tumor lung cancer Rb1 (13q) transcription retinoblastoma small-cell lung carcinoma p53 (17p) transcription Li-Fraumeni breast, colon, syndrome & lung cancer BRCA1(17q) transcriptional breast cancer breast/ovarian tumors BRCA2 (13q) regulator/DNA repair  p53 TUMOR SUPPRESSOR GENES ⚫ Phosphyorylated p53 activates transcription of p21 gene ⚫ Cell cycle arrested to allow DNA to be repaired ⚫ If damage cannot be repaired --> cell death (apoptosis) ⚫ Disruption/deletion of p53 gene ⚫ Inactivation of p53 protein --> uncorrected DNA damage --> uncontrolled cell proliferation --> cancer Cancer treatment/therapies Promising Cancer targets  Apoptosis – Programmed Cell Death (True/False) In adult tissues cell death exactly balances cell division In apoptosis the cell destroys itself from within and avoids leakage of the cell contents into the extracellular space. Why do you think that this occurs via a different mechanism than in necrosis? What are some signals that indicate to a cell that apoptosis needs to occur? Where do these signals come from? What are some cellular components involved in the apoptotic pathway? What is the difference between a mitogen, a growth factor, and a survival factor? In what phase of the cell cycle do cells exit to undergo apoptosis? What effects do telomeres and telomerase have on cell aging and death? If you could turn on telomerase activity in all of our cells, would it prevent aging? Do the following types of cells exist in humans? – Cells that do not grow and do not divide – Cells that grow, but do not divide – Cells that divide, but do not grow – Cells that grow and divide