Mechanisms of Cell Death

Mechanisms of Cell Death CELL DEATH AND FORMATION OF THE SEMICIRCULAR CANALS Carol M. Troy, MD PhD August 24, 2009 FROM: Fekete et al., Development 124: 2451 (1997) PHENOMENOLOGY OF CELL DEATH I. DEVELOPMENT A. MORPHOGENESIS: SCULPTING/SHAPING STRUCTURES CREATION OF CAVITIES AND TUBES FUSION OF TISSUE MASSES (PALATE/NEURAL TUBE) CREATION OF FORM (DIGITS) PHENOMENOLOGY OF CELL DEATH: DEVELOPMENT B. DELETION OF UNNEEDED STRUCTURES KIDNEY: PRONEPHROS AND MESONEPHROS BRAIN: CORTICAL SUBPLATE NEURONS UROGENITAL SYSTEM: WOLFFIAN AND MÜLLERIAN DUCTS CELL DEATH AND FORMATION OF DIGITS PHENOMENOLOGY OF CELL DEATH: DEVELOPMENT C. ELIMINATION OF ECTOPIC, DAMAGED OR UNEEDED CELLS CELLS WITH DNA DAMAGE IMMUNE SYSTEM CELLS ECTOPIC CELLS FROM: Chen and Zhao, J. Exp. Zool. 282:691 (1998). 1

PHENOMENOLOGY OF CELL DEATH: DEVELOPMENT NEURONAL CULLING AS REGULATED BY COMPETITION FOR TARGET-SUPPLIED TROPHIC FACTOR D. CULLING: REGULATION OF CELL NUMBERS NERVOUS SYSTEM: MATCHING NEURONS WITH TARGETS MATCHING SCHWANN CELL AND OLIGODENDROCYTES WITH AXONS NORMAL DEVELOPMENTAL NEURONAL DEATH IS REGULATED BY TARGET DERIVED TROPHIC FACTORS NEURONAL CULLING AS REGULATED BY COMPETITION FOR TARGET-SUPPLIED TROPHIC FACTOR NEURON NUMBER IN CHICK ION 25,000 20,000 15,000 NORMAL 10,000 5,000 ENUCLEATED 0 36 38 40 42 44 46 P2 P4 DEVELOPMENTAL STAGE Clarke, Rogers & Cowan J. Comp. Neurol. 167: 125 (1976) PHENOMENOLOGY OF CELL DEATH II. ELIMINATION OF CELLS WITH DNA DAMAGE III. DEFENSE FROM PATHOGENS IV. REGULATION OF CELL NUMBERS HOMEOSTASIS OF ORGAN/TISSUE SIZE PREVENTION OF UNREGULATED CELL GROWTH IMMUNE CELL NUMBERS 2

Cell death maintains the mature organism PROPAGATION SIGNALING EVENTS TRANSCRIPTIONAL POST-TRANSCRIPTIONAL POST-TRANSLATIONAL In the course of a year your body weight in cells should die. DYSREGULATION OF CELL DEATH: DISEASE A. CANCER EXECUTION CASPASES - DEATH PROTEASES B. HYPOXIC/ANOXIC CELL DEATH - Brain, heart C. NEURODEGENERATIVE DISORDERS - AD, PD D. ACUTE AND CHRONIC RENAL FAILURE E. VIRAL PATHOGENESIS INITIATION DEATH STIMULI CELL DEATH 3

CASPASES - 2: EVIDENCE FOR ROLE IN APOPTOSIS OVER-EXPRESSION CAUSES APOPTOTIC DEATH ACTIVATED IN DYING CELLS CLEAVED FORMS DETECTABLE BY WESTERN & ANTIBODIES CAN MEASURE ACTIVITY IN DYING CELL EXTRACTS WITH SUBSTRATES cytochrome c-4cys red phosphatidylserine, Blue histone H2B coupled to GFP Goldstein et al. Cell Death and Differentiation (2005) 12, 453 462. BLOCK APOPTOTIC DEATH WITH CASPASE INHIBITORS REVERSIBLE AND IRREVERSIBLE PSEUDOSUBSTRATE PEPTIDES (zdevd-fmk) MOLECULAR INHIBITION: ANTISENSE, sirna VIRAL INHIBITORS: CRMA, p35 IAPs NULL ANIMALS SHOW DEFECTIVE CELL DEATH APOPTOTIC DEATH vs NECROTIC DEATH EMBRYOGENIC DEFECTS IN A MOUSE LACKING CASPASE-9 PRESENT IN DEVELOPING TISSUES CYTOPLASMIC BLEBBING CELLULAR & NUCLEAR PYKNOSIS RESPONSE TO CELL INJURY, TOXINS CELL & NUCLEAR SWELLING CHROMATIN CONDENSATION DNA DEGRADATION BY ENDONUCLEASES (FORMATION OF DNA LADDER) FORMATION OF MEMBRANE-LIMITED APOPTOTIC BODIES RANDOM DNA DEGRADATION LOSS OF MEMBRANE INTEGRITY & LOSS OF CYTOPLASMIC CONTENTS PHAGOCYTOSIS OF APOPTOTIC BODIES ABSENCE OF INFLAMMATORY RESPONSE INFLAMMATORY RESPONSE Kerr, Wylie and Currie From: Kuida et al Cell:94: 325-337, 1998 CASPASES - 1: PROPERTIES EXECUTORS OF APOPTOTIC DEATH CYSTEINE PROTEASES CLEAVE AFTER ASP - ARE ASPARTASES WHEN ACTIVATED, CLEAVE CELLULAR SUBSTRATES, LEADING TO APOPTOTIC DEATH CASPASES: HOW ARE THEY ACTIVATED? CONSTITUTIVELY EXPRESSED IN CELLS AS INACTIVE PRO-FORMS TWO GENERAL CLASSES OF CASPASES: INITIATOR AND EFFECTOR OR EXECUTIONER CAPASES DIFFERENT CASPASES DIFFER IN SPECIFICITY, MEANS OF ACTIVATION, AND SUBCELLULAR COMPARTMENTALIZATION 4

Phylogenetic Relations of Caspases Cell Death and DifferentiationLamkanfi et al. (2002) 9: 358-361 CASPASES: ACTIVATION OF INITIATOR CASPASES BY INDUCED PROXIMITY CASPASE - DEPENDENT ACTIVATION OF THE CAD ENDONUCLEASE (INACTIVE CED3, CASP 2,8,9,10) PRO DOMAIN P20 P10 APOPTOTIC STIMULUS APOPTOTIC STIMULUS CASPASE CAD ICAD CAD Activating platform Activating platform Autocleavage (ACTIVE) (ACTIVE) CASPASES: ACTIVATION OF EFFECTOR CASPASES BY CLEAVAGE (INACTIVE CASP 3,6,7) PRO DOMAIN P20 P10 D1 D2 APOPTOTIC STIMULUS INITIATOR CASPASES PRO P20 P10 (ACTIVE) 5

Caspase Regulation VERTEBRATE PRO-APOPTOTIC FAMILY MEMBERS zymogen OVER-EXPRESSION OF PRO-APOPTOTIC FAMILY MEMBERS PROMOTES APOPTOSIS CELLS OF ANIMALS NULL FOR PRO-APOPTOTIC MEMBERS SHOW LESS SUCEPTIBILITY TO APOPTOTIC DEATH Diablo CAN FORM HETERODIMERS WITH ANTI-APOPTOTIC FAMILY MEMBERS VIA BH DOMAINS AS WELL AS WITH ONE ANOTHER HtrA2 ABILITY TO BIND FAMILY MEMBERS ESSENTIAL FOR PRO-APOPTOTIC ACTIVITY casp1/2 MITOCHONDRIA AND ACTIVATION OF APAF1 IN MAMMALIAN CELLS mitochondrion APAF1 CASPASE-9 ACTIVATION CASPASE-9 CASP 3,6,7 Bcl2 Proteins MITOCHONDRIA AND ACTIVATION OF APAF1 IN MAMMALIAN CELLS WHERE DO PRO-AND ANTI-APOPTOTIC MEMBERS FIT INTO THIS SCHEME?? mitochondrion APAF1 (APOPTOSOME) CASPASE ACTIVATION CASPASE 9 CASP 3,6,7 6

THE FAMILY AND RELEASE FROM MITOCHONDRIA BOTH AND ARE REQUIRED FOR MITOCHONDRIALLY-DEPENDENT APOPTOSIS ANTI-APOPTOTIC MEMBERS ARE RESIDENT IN MITOCHONDRIA OVER-EXPRESSION OF ANTI-APOPTOTIC MEMBERS (eg AND BCLXL ) BLOCKS RELEASE AND APOPTOSIS OVER-EXPRESSION OF PRO-APOPTOTIC AND MEMBERS CAUSES RELEASE & DEATH BOTH ARE BH1-3 FAMILY MEMBERS AND BOTH APPEAR TO BE REQUIRED FOR MANY CASES OF APOPTOSIS KNOCKOUT OF AND PROTECTS CELLS FROM DEATH MITOCHONDRIA AND APOPTOTIC DEATH -2 AND AND APOPTOTIC DEATH mitochondrion APAF1 (APOPTOSOME) mitochondrion APAF1 (APOPTOSOME) CASPASE 3,6,7 ACTIVATION CASPASE 3,6,7 ACTIVATION HOW DO PRO-APOPTOTIC FAMILY MEMBERS PROMOTE APOPTOSIS? HOW DO BH3-ONLY MOLECULES CONTRIBUTE TO MITOCHONDRIAL-DEPENDENT APOPTOSIS? MITOCHONDRIAL-DEPENDENT APOPTOSIS REQUIRES BH3-ONLY PROTEINS AS WELL AS / THERE APPEARS TO BE A TWO STEP MECHANISM REQUIRING BOTH FAMILY MEMBERS AND BH3-DOMAIN ONLY FAMILY MEMBERS OVER-EXPRESSION OF BH3-ONLY PROTEINS INDUCES APOPTOSIS, BUT NOT IN ABSENCE OF OR. SO THE LATTER APPEAR TO WORK DOWNSTREAM OF BH3 MOLECULES IN ABSENCE OF BH3-ONLY PROTEINS AND DO NOT CHANGE CONFORMATION AND FORM PORES THUS, APOPTOTIC DEATH VIA THE MITOCHONDRION REQUIRES BOTH BH3-ONLY PROTEINS AND / 7

HOW DO / AND BH3-ONLY PROTEINS COOPERATE TO INDUCE CYTOCHROME-C RELEASE AND APOPTOSIS? ADDITIONAL REGULATORS OF CELL DEATH 1) BH3-ONLY PROTEINS DISPLACE / FROM AND OTHER ANTI-APOPTOTIC FAMILY MEMBERS IAPS - INHIBITOR OF APOPTOSIS PROTEINS HOW DO BH3-ONLY PROTEINS COOPERATE WITH / TO INDUCE CYTOCHROME-C RELEASE AND APOPTOSIS? Inhibitor of Apoptosis Proteins APOPTOTIC STIMULI BH3-ONLY PROTEIN CYT-C CYT-C PORE FORMATION IN MITOCHONDRIAL OUTER MEMBRANE RELEASE OF Verhagen et al. Genome Biol. 2:3009.1-10, 2001 THE RHEOSTAT MODEL OF CELL DEATH IAPS BH3- ONLY SURVIVAL FAMILY OF PROTEINS THAT INHIBIT CASPASES (3,7,9) MULTIPLE FAMILY MEMBERS IN MAMMALS. ALSO PRESENT IN INSECTS AND VIRUSES. BH3- ONLY BH3- ONLY BH3- BH3- ONLY ONLY BH3- ONLY BH3- ONLY BH3- ONLY DEATH ALL HAVE BIR (BACULOVIRAL IAP REPEAT) DOMAINS THAT ARE REQUIRED FOR BINDING AND INHIBITING CASPASES SEVERAL (IAP1,2 AND XIAP) HAVE RING FINGERS AND E3 LIGASE ACTIVITY AND CAN LEAD TO DEGRADATION OF CASPASES AND OTHER PRO-APOPTOTIC MOLECULES 8

IAPS MAY FUNCTION AS A CHECK POINT BEFORE THE LAST IRREVERSIBLE STAGE OF DEATH LEVELS CAN BE UP-REGULATED BY GROWTH FACTORS (EG., GDNF) OR DOWN-REGULATED BY APOPTOTIC SIGNALS OVER-EXPRESSED IN SOME TUMORS - SO POTENTIAL CLINICAL TARGET SMAC/DIABLO INHIBITS IAPS BIM BIM mitochondrion APAF1 CASPASE 3, 7 ACTIVATION IAPs SMAC/DIABLO X IAPS INHIBIT CASPASES AND APOPTOTIC DEATH SMAC DISPLACES IAPS FROM CASPASES BIM BIM mitochondrion N-TERMINAL TETRA-PEPTIDE OF SMAC APAF1 IAPs CASPASE 3,7 ACTIVATION ciap1,2 XIAP X XIAP BIR3 N-TERMINAL TETRA-PEPTIDE OF CASPASE 9 SMALL SUBUNIT ADDITIONAL REGULATORS OF CELL DEATH SMAC/DIABLO SMAC/DIABLO - INHIBITORS OF IAPS RELEASED FROM MITOCHONDRIA BY BLOCKS IAPS FROM INHIBITING CASPASES: PRO-APOPTOTIC REQUIRED FOR DEATH IN AT LEAST SOME PARADIGMS. IN OTHERS, IT MAKES CELLS MORE SUCEPTIBLE TO DEATH 9

OMI/HTRA2 INHIBITS IAPS THE RECEPTOR-MEDIATED PATHWAY OF APOPTOTIC DEATH DEATH DOMAIN BIM BIM mitochondrion OMI/HTRA2 APAF1 CASPASE 3,7 ACTIVATION X IAPs SER/THR PROTEASE TRAIL-R1 TRAIL-R2 CELL INTERIOR OMI/HTRA2 DEATH PROMOTING RECEPTORS AND LIGANDS RELEASED FROM MITOCHONDRIA BY IS A SERINE/THREONINE PROTEASE DEGRADES IAPS: PRO-APOPTOTIC MAKES CELLS MORE SUCEPTIBLE TO DEATH UP-REGULATED BY P53 LIGAND TNFα FAS ligand TRAIL RECEPTOR TNFαR1 FAS TRAIL-R(DR-4 & DR-5) DEATH BY MURDER - RECEPTOR MEDIATED IN ADDITION TO SUICIDE (THE INTRINSIC APOPTOTIC MECHANISM), THERE IS ALSO A MAMMALIAN MECHANISM FOR MURDERING CELLS (THE EXTRINSIC APOPTOTIC PATHWAY) THE EXTRINSIC APOPTOTIC PATHWAY IS REGULATED BY A SERIES OF SPECIFIC DEATH-PROMOTING RECEPTORS AND LIGANDS. OCCUPATION OF THESE RECEPTORS BRINGS ABOUT ACTIVATION OF PATHWAYS THAT CULMINATE IN CELL DEATH. THE RECEPTOR-MEDIATED PATHWAY OF APOPTOTIC DEATH = DD = DED TNFα TNFR1 FADD CASPASES 8,10 BID tbid MITOCHONDRIAL PATHWAY CASPASE 3 10

DEATH PROMOTING RECEPTORS AND LIGANDS-2 THE EXTRINSIC DEATH PATHWAY BEGINS WITH RECEPTOR-MEDIATED ACTIVATION OF INITIATOR CASPASES 8 AND/0R 10 TRANSCRIPTIONAL TARGETS OF P53 IN APOPTOSIS (DNA DAMAGE) P53 PHOSPHORYLATION AND STABILIZATION E2F THE PATHWAY CAN EITHER BYPASS MITOCHONDRIAL INVOLVEMENT IN DEATH OR CAN INVOLVE MITOCHONDRIA AS A MEANS OF AMPLIFICATION THE SAME CELL CAN EXPRESS BOTH DEATH RECEPTOR AND DEATH LIGAND SIAH JNK/JUN BIM p53 BID PUMA NOXA PAC1 MAPK Cell specific Stimulus specific SUSCEPTIBILITY IS SUBJECT TO REGULATION BY PATHWAY ANTAGONISTS APAF-1 CASPASE 6 DEATH PROMOTING RECEPTORS AND LIGANDS-3 THE EXTRINSIC PATHWAY CONTRIBUTES TO DEATH IN A VARIETY OF CONTEXTS MANY TUMOR CELLS EXPRESS EXTRINSIC PATHWAY RECEPTORS, BUT ALSO OVER-EXPRESS EXPRESS PATHWAY ANTAGONISTS (50% of colon cancers have amplified DcR3 gene) EXPRESSION OF LIGAND AND RECEPTORS AS WELL AS OF PATHWAY ANTAGONISTS IS SUBJECT TO REGULATION FAS AND THE TRANSCRIPTIONAL REGULATION OF EXTRINSIC DEATH PATHWAY FAS-L p53 cjun FKH E2F NFKB FAS BIM BIM mitochondrion SMAC/DIABLO AIF APAF1 CASPASE 3,7 ACTIVATION IAPs ROLE OF TRANSCRIPTION IN APOPTOSIS WHAT SIGNALS KEEP CELL FROM DYING? ACTIVATION OF AKT/PKB Growth factor IN MANY, BUT NOT ALL PARADIGMS OF APOPTOTIC DEATH CELLS MUST SYNTHESIZE SPECIFIC GENES TO DIE THE PATHWAYS THAT REGULATE SUCH DEATH-ASSOCIATED ASSOCIATED GENES APPEAR TO BE ACTIVATED BY MECHANISMS THAT ARE INDEPENDENT OF MITOCHONDRIA. THE GENE PRODUCTS OF THESE PATHWAYS CAN ACT BOTH UPSTREAM AND DOWNSTREAM OF MITOCHONDRIA P- BH3-DOMAIN ONLY MOLECULES SEEM TO BE COMMON GENE TARGETS (eg. BIM IS REGULATED BY JUN, E2F, AND FORKHEAD) 11

AKT BLOCKS DEATH AT MULTIPLE LEVELS OF THE APOPTOTIC MECHANISM MECHANISMS OF APOPTOSIS RESISTANCE-2 FAS-L p53 cjun FKH E2F NFKB FAS BCLX-L BAD BIM BIM mitochondrion SMAC AIF APAF1 CASPASE 3,6,7 ACTIVATION IAPs BLOCK APOPTOTIC TRANSC. PATHWAYS (FKH PHOSPHORYLATION) ELEVATE ANTI-APOPTOTICAPOPTOTIC MOLECULES PHOSPHORYLATE, EXCLUDE PRO- APOPTOTIC BAD INCREASED EXPRESSION OF IAPS -SURVIVIN IN NEUROBLASTOMA -ciap1/2 IN LUNG CA -ciap1 IN ESOPHAGEAL SQUAMOUS CELL CA -ciap1 INCREASES RESISTANCE TO CHEMOTHERAPY -XIAP IN OVARIAN CA PHENOMENOLOGY OF CELL DEATH V. DISEASE A. CANCER B. HYPOXIC/ANOXIC CELL DEATH - Brain, heart C. NEURODEGENERATIVE DISORDERS - AD, PD D. ACUTE AND CHRONIC RENAL FAILURE E. VIRAL PATHOGENESIS MECHANISMS OF APOPTOSIS RESISTANCE-3 INCREASED EXPRESSION OF IN ALL, AML, CLL,MULTIPLE MYELOMA, PROSTATE CA, NEUROBLASTOMA DECREASED EXPRESSION OF IN COLON CA, BREAST CA MECHANISMS OF APOPTOSIS RESISTANCE-1 MUTATIONS OF CASPASES -MCF7 BREAST CA HAS NO CASPASE-3 EXPRESSION -DECREASED CASPASE-7 EXPRESSION IN COLON CA -HYPERMETHYLATION OF CASPASE-8 PROMOTER LOSS OF APAF-1 EXPRESSION IN MELANOMA STRATEGIES FOR TARGETING CANCER INHIBITION OF OVEREXPRESSED ANTI- APOPTOTIC MOLECULES -e.g., BH3 PROTEINS, SURVIVIN, OTHER IAPS ENHANCE RECEPTOR MEDIATED DEATH VIA TRAIL-R ENHANCE PRO-APOPTOTIC PATHWAYS -small molecule mimetic of SMAC/Diablo 12

MAINTENANCE OF HOMEOSTASIS TIGHT REGULATION OF DEATH PATHWAYS AT SEVERAL LEVELS IS ESSENTIAL A BETTER UNDERSTANDING OF THE MOLECULAR MECHANISM(S) OF APOPTOSIS WILL ENABLE DESIGN OF TARGETED THERAPIES FOR DISORDERS WITH DYSREGULATED CELL DEATH. 13