Mechanisms of Cell Death Carol M. Troy, MD PhD August 24, 2009 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) 1
CELL DEATH AND FORMATION OF DIGITS FROM: Chen and Zhao, J. Exp. Zool. 282:691 (1998). CELL DEATH AND FORMATION OF THE SEMICIRCULAR CANALS FROM: Fekete et al., Development 124: 2451 (1997) 2
PHENOMENOLOGY OF CELL DEATH: DEVELOPMENT B. DELETION OF UNNEEDED STRUCTURES KIDNEY: PRONEPHROS AND MESONEPHROS BRAIN: CORTICAL SUBPLATE NEURONS UROGENITAL SYSTEM: WOLFFIAN AND MÜLLERIAN DUCTS PHENOMENOLOGY OF CELL DEATH: DEVELOPMENT C. ELIMINATION OF ECTOPIC, DAMAGED OR UNEEDED CELLS CELLS WITH DNA DAMAGE IMMUNE SYSTEM CELLS ECTOPIC CELLS 3
PHENOMENOLOGY OF CELL DEATH: DEVELOPMENT 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 ION 25,000 NEURON NUMBER IN CHICK 20,000 15,000 10,000 5,000 0 NORMAL ENUCLEATED 36 38 40 42 44 46 P2 P4 DEVELOPMENTAL STAGE Clarke, Rogers & Cowan J. Comp. Neurol. 167: 125 (1976) 4
NEURONAL CULLING AS REGULATED BY COMPETITION FOR TARGET-SUPPLIED TROPHIC FACTOR 5
NEURONAL CULLING AS REGULATED BY COMPETITION FOR TARGET-SUPPLIED TROPHIC FACTOR 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 6
Cell death maintains the mature organism In the course of a year your body weight in cells should die. DYSREGULATION OF CELL DEATH: DISEASE A. CANCER B. HYPOXIC/ANOXIC CELL DEATH - Brain, heart C. NEURODEGENERATIVE DISORDERS - AD, PD D. ACUTE AND CHRONIC RENAL FAILURE E. VIRAL PATHOGENESIS 7
INITIATION DEATH STIMULI PROPAGATION SIGNALING EVENTS TRANSCRIPTIONAL POST-TRANSCRIPTIONAL POST-TRANSLATIONALTRANSLATIONAL 8
EXECUTION CASPASES - DEATH PROTEASES CELL DEATH 9
cytochrome c-4cys red phosphatidylserine, Blue histone H2B coupled to GFP Goldstein et al. Cell Death and Differentiation (2005) 12, 453 462. APOPTOTIC DEATH vs NECROTIC DEATH PRESENT IN DEVELOPING TISSUES RESPONSE TO CELL INJURY, TOXINS CYTOPLASMIC BLEBBING CELLULAR & NUCLEAR PYKNOSIS 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 10
CASPASES - 1: PROPERTIES EXECUTORS OF APOPTOTIC DEATH CYSTEINE PROTEASES CLEAVE AFTER ASP - ARE ASPARTASES WHEN ACTIVATED, CLEAVE CELLULAR SUBSTRATES, LEADING TO APOPTOTIC DEATH DIFFERENT CASPASES DIFFER IN SPECIFICITY, MEANS OF ACTIVATION, AND SUBCELLULAR COMPARTMENTALIZATION 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 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 11
EMBRYOGENIC DEFECTS IN A MOUSE LACKING CASPASE-9 From: Kuida et al Cell:94: 325-337, 1998 CASPASES: HOW ARE THEY ACTIVATED? CONSTITUTIVELY EXPRESSED IN CELLS AS INACTIVE PRO-FORMS TWO GENERAL CLASSES OF CASPASES: INITIATOR AND EFFECTOR OR EXECUTIONER CAPASES 12
Phylogenetic Relations of Caspases Cell Death and DifferentiationLamkanfi et al. (2002) 9: 358-361 CASPASES: ACTIVATION OF INITIATOR CASPASES BY INDUCED PROXIMITY (INACTIVE CED3, CASP 2,8,9,10) PRO DOMAIN P20 P10 CARD APOPTOTIC STIMULUS CARD Activating CARD platform CARD CARD CARD CARD Activating platform CARD CARD Autocleavage (ACTIVE) (ACTIVE) 13
CASPASES: ACTIVATION OF EFFECTOR CASPASES BY CLEAVAGE (INACTIVE CASP 3,6,7) PRO DOMAIN P20 P10 PRO D1 D2 APOPTOTIC STIMULUS INITIATOR CASPASES P20 P10 (ACTIVE) 14
CASPASE - DEPENDENT ACTIVATION OF THE CAD ENDONUCLEASE APOPTOTIC STIMULUS CASPASE CAD ICAD CAD 15
Caspase Regulation zymogen Diablo HtrA2 casp1/2 16
Bcl2 Proteins VERTEBRATE PRO-APOPTOTIC BCL2 FAMILY MEMBERS OVER-EXPRESSION OF PRO-APOPTOTIC FAMILY MEMBERS PROMOTES APOPTOSIS CELLS OF ANIMALS NULL FOR PRO-APOPTOTIC MEMBERS SHOW LESS SUCEPTIBILITY TO APOPTOTIC DEATH CAN FORM HETERODIMERS WITH ANTI-APOPTOTIC FAMILY MEMBERS VIA BH DOMAINS AS WELL AS WITH ONE ANOTHER ABILITY TO BIND BCL2 FAMILY MEMBERS ESSENTIAL FOR PRO-APOPTOTIC ACTIVITY 17
MITOCHONDRIA AND ACTIVATION OF APAF1 IN MAMMALIAN CELLS APOPTOTIC STIMULI mitochondrion CYTOCHROME C APAF1 CASPASE-9 ACTIVATION CASPASE-9 CASP 3,6,7 MITOCHONDRIA AND ACTIVATION OF APAF1 IN MAMMALIAN CELLS APOPTOTIC STIMULI mitochondrion WHERE DO PRO-AND ANTI-APOPTOTIC APOPTOTIC BCL2 MEMBERS FIT INTO THIS SCHEME?? CYTOCHROME C APAF1 (APOPTOSOME) CASPASE ACTIVATION CASPASE 9 CASP 3,6,7 18
THE BCL2 FAMILY AND CYTOCHROME C RELEASE FROM MITOCHONDRIA ANTI-APOPTOTIC BCL2 MEMBERS ARE RESIDENT IN MITOCHONDRIA OVER-EXPRESSION EXPRESSION OF ANTI-APOPTOTIC APOPTOTIC BCL2 MEMBERS (eg BCL2 AND BCLXL ) BLOCKS CYTOCHROME C RELEASE AND APOPTOSIS MITOCHONDRIA AND APOPTOTIC DEATH -2 mitochondrion BCL2 BCL2 CYTOCHROME C APAF1 (APOPTOSOME) CASPASE 9 ACTIVATION CASPASE 3,6,7 ACTIVATION 19
HOW DO PRO-APOPTOTIC BCL2 FAMILY MEMBERS PROMOTE APOPTOSIS? THERE APPEARS TO BE A TWO STEP MECHANISM REQUIRING BOTH BAX FAMILY MEMBERS AND BH3-DOMAIN ONLY FAMILY MEMBERS BOTH BAX AND BAK ARE REQUIRED FOR MITOCHONDRIALLY-DEPENDENT APOPTOSIS OVER-EXPRESSION OF PRO-APOPTOTIC BAX AND BAK MEMBERS CAUSES CYTOCHROME C RELEASE & DEATH BOTH ARE BH1-3 BAX FAMILY MEMBERS AND BOTH APPEAR TO BE REQUIRED FOR MANY CASES OF APOPTOSIS KNOCKOUT OF BAX AND BAK PROTECTS CELLS FROM DEATH 20
BAX AND BAK AND APOPTOTIC DEATH APOPTOTIC STIMULI BAX BAX mitochondrion BCL2 BCL2 BAX BAK BAX BAK CYTOCHROME C APAF1 (APOPTOSOME) CASPASE 9 ACTIVATION CASPASE 3,6,7 ACTIVATION HOW DO BH3-ONLY MOLECULES CONTRIBUTE TO MITOCHONDRIAL-DEPENDENT APOPTOSIS? MITOCHONDRIAL-DEPENDENT APOPTOSIS REQUIRES BH3-ONLY PROTEINS AS WELL AS BAX/BAK OVER-EXPRESSION OF BH3-ONLY PROTEINS INDUCES APOPTOSIS, BUT NOT IN ABSENCE OF BAX OR BAK. SO THE LATTER APPEAR TO WORK DOWNSTREAM OF BH3 MOLECULES IN ABSENCE OF BH3-ONLY PROTEINS BAX AND BAK DO NOT CHANGE CONFORMATION AND FORM PORES THUS, APOPTOTIC DEATH VIA THE MITOCHONDRION REQUIRES BOTH BH3-ONLY PROTEINS AND BAX/BAK 21
HOW DO BAX/BAK AND BH3-ONLY PROTEINS COOPERATE TO INDUCE CYTOCHROME-C RELEASE AND APOPTOSIS? 1) BH3-ONLY PROTEINS DISPLACE BAX/BAK FROM BCL2 AND OTHER ANTI-APOPTOTIC FAMILY MEMBERS HOW DO BH3-ONLY PROTEINS COOPERATE WITH BAX/BAK TO INDUCE CYTOCHROME-C RELEASE AND APOPTOSIS? BCL2 BAK BCL2 BAX APOPTOTIC STIMULI BH3-ONLY PROTEIN BCL2BCL2 BAK BAX BAK BAX BAX BAK BAK BAX BAX BAK CYT-C CYT-C C PORE FORMATION IN MITOCHONDRIAL OUTER MEMBRANE RELEASE OF CYTOCHROME C 22
THE RHEOSTAT MODEL OF CELL DEATH BAK BCL2 BCL2 BH3- BCL2 ONLY SURVIVAL BAX BCL2 BCL2 BH3- ONLY BH3- BCL2 BH3- ONLY BCL2 ONLY BH3- BCL2 ONLY BH3- ONLY BCL2 BCL2 BH3- ONLY BH3- ONLY BAX BAK DEATH ADDITIONAL REGULATORS OF CELL DEATH IAPS - INHIBITOR OF APOPTOSIS PROTEINS 23
Inhibitor of Apoptosis Proteins Verhagen et al. Genome Biol. 2:3009.1-10, 2001 IAPS FAMILY OF PROTEINS THAT INHIBIT CASPASES (3,7,9) MULTIPLE FAMILY MEMBERS IN MAMMALS. ALSO PRESENT IN INSECTS AND VIRUSES. 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 24
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 IAPS INHIBIT CASPASES AND APOPTOTIC DEATH APOPTOTIC STIMULI BIM BIM BCL2mitochondrion BAX BAX CASPASE 9 ACTIVATION CYTOCHROME C APAF1 IAPs CASPASE 3,7 ACTIVATION ciap1,2 XIAP X 25
ADDITIONAL REGULATORS OF CELL DEATH SMAC/DIABLO - INHIBITORS OF IAPS SMAC/DIABLO INHIBITS IAPS APOPTOTIC STIMULI BIM BIM BCL2mitochondrion BAX BAX SMAC/DIABLO CYTOCHROME C APAF1 CASPASE 9 ACTIVATION CASPASE 3, 7 ACTIVATION IAPs X 26
SMAC DISPLACES IAPS FROM CASPASES XIAP BIR3 N-TERMINAL TETRA-PEPTIDE OF SMAC N-TERMINAL TETRA-PEPTIDE OF CASPASE 9 SMALL SUBUNIT SMAC/DIABLO RELEASED FROM MITOCHONDRIA BY APOPTOTIC STIMULI BLOCKS IAPS FROM INHIBITING CASPASES: PRO-APOPTOTIC REQUIRED FOR DEATH IN AT LEAST SOME PARADIGMS. IN OTHERS, IT MAKES CELLS MORE SUCEPTIBLE TO DEATH 27
OMI/HTRA2 INHIBITS IAPS APOPTOTIC STIMULI BIM BIM BCL2mitochondrion BAX BAX OMI/HTRA2 CYTOCHROME C APAF1 CASPASE 9 ACTIVATION CASPASE 3,7 ACTIVATION X IAPs SER/THR PROTEASE OMI/HTRA2 RELEASED FROM MITOCHONDRIA BY APOPTOTIC STIMULI IS A SERINE/THREONINE PROTEASE DEGRADES IAPS: PRO-APOPTOTIC MAKES CELLS MORE SUCEPTIBLE TO DEATH UP-REGULATED BY P53 28
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 DEATH DOMAIN TRAIL-R1 TRAIL-R2 CELL INTERIOR 29
DEATH PROMOTING RECEPTORS AND LIGANDS LIGAND TNFα FAS ligand TRAIL RECEPTOR TNFαR1 FAS TRAIL-R(DR-4 & DR-5) THE RECEPTOR-MEDIATED PATHWAY OF APOPTOTIC DEATH = DD TNFα = DED TNFR1 FADD CASPASES 8,10 BID tbid BAX MITOCHONDRIAL PATHWAY CASPASE 3 30
DEATH PROMOTING RECEPTORS AND LIGANDS-2 THE EXTRINSIC DEATH PATHWAY BEGINS WITH RECEPTOR-MEDIATED ACTIVATION OF INITIATOR CASPASES 8 AND/0R 10 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 SUSCEPTIBILITY IS SUBJECT TO REGULATION BY PATHWAY ANTAGONISTS 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 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 31
ROLE OF TRANSCRIPTION IN APOPTOSIS IN MANY, BUT NOT ALL PARADIGMS OF APOPTOTIC DEATH CELLS MUST SYNTHESIZE SPECIFIC GENES TO DIE THE PATHWAYS THAT REGULATE SUCH DEATH-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 BH3-DOMAIN ONLY MOLECULES SEEM TO BE COMMON GENE TARGETS (eg. BIM IS REGULATED BY JUN, E2F, AND FORKHEAD) TRANSCRIPTIONAL TARGETS OF P53 IN APOPTOSIS APOPTOTIC STIMULI (DNA DAMAGE) P53 PHOSPHORYLATION AND STABILIZATION E2F SIAH p53 PAC1 MAPK JNK/JUN BIM BAX BID PUMA NOXA APAF-1 CASPASE 6 Cell specific Stimulus specific 32
FAS AND THE TRANSCRIPTIONAL REGULATION OF EXTRINSIC DEATH PATHWAY APOPTOTIC STIMULI FAS-L p53 cjun FKH E2F NFKB FAS BIM BIM BCL2mitochondrion BCL2 BAX BAX AIF CYTOCHROME C APAF1 SMAC/DIABLO CASPASE 9 ACTIVATION CASPASE 3,7 ACTIVATION IAPs WHAT SIGNALS KEEP CELL FROM DYING? ACTIVATION OF AKT/PKB Growth factor P- 33
AKT BLOCKS DEATH AT MULTIPLE LEVELS OF THE APOPTOTIC MECHANISM APOPTOTIC STIMULI FAS-L p53 cjun FKH E2F NFKB FAS BLOCK APOPTOTIC TRANSC. PATHWAYS (FKH PHOSPHORYLATION) BAD BCLX-L BIM BIM BCL2mitochondrion SMAC BCL2 BAX BAX AIF CYTOCHROME C APAF1 ELEVATE ANTI-APOPTOTIC MOLECULES CASPASE 9 ACTIVATION CASPASE 3,6,7 ACTIVATION IAPs PHOSPHORYLATE, EXCLUDE PRO- APOPTOTIC BAD 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 34
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 MECHANISMS OF APOPTOSIS RESISTANCE-2 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 35
MECHANISMS OF APOPTOSIS RESISTANCE-3 INCREASED EXPRESSION OF BCL2 IN ALL, AML, CLL,MULTIPLE MYELOMA, PROSTATE CA, NEUROBLASTOMA DECREASED EXPRESSION OF BAX IN COLON CA, BREAST CA STRATEGIES FOR TARGETING CANCER INHIBITION OF OVEREXPRESSED ANTI- APOPTOTIC MOLECULES -e.g. BCL2, BH3 PROTEINS, SURVIVIN, OTHER IAPS ENHANCE RECEPTOR MEDIATED DEATH VIA TRAIL-R ENHANCE PRO-APOPTOTIC PATHWAYS -small molecule mimetic of SMAC/Diablo 36
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. 37