Apoptosis:Death Comes

for the Cell

Joe W. Ramosjoeramos@hawaii.edu

From Ingmar Bergman�s The Seventh Seal

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Mutations in proteins that regulate cell proliferation, survival and death

can contribute to oncogenesis

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The Chessboard

Ways to Die

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From Okada and Mak, Nat. Rev. Cancer 4:592-603

The Morphology of Death

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Apoptosis: Programmed Cell Death

A term used to describe the morphological changes associated with programmed cell death.

The term was originally used by Wyllie and his colleagues and is from the Greek meaning �dropping away� as the leaves from a tree.

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Apoptosis

l Active cell deathl Requires energy and RNA and protein

synthesisl Characteristic morphological featuresl DNA cleaved, chromatin condensesl Cells shrinkl Formation of apoptotic bodyl Cleared by phagocytosisl No inflammation=no tissue damage 7

Necrosisl Passive cell deathl Cells swell upl Membrane breaks down and cellular contents

leak outl Nucleus disintegratesl Cell ghostsl Inflammatory=tissue damage

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The function of Cell death

l Multicellular developmentl involved in deletion of entire structures, l sculpting of tissues, l and regulates the neuron number

l The immune responsel The body�s defense against

cancer

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Death and the mouse�s paw

Fig 18-18

Dark Green fluorescence indicates apoptotic cells.

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Apoptosis regulates nerve cell targeting

Fig 18-2011

Apoptosis in Lymphocyte development

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How do we recognize Programmed Cell Death?

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The Face of Cell Death: Apoptosis

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Detection of apoptotic cells

l Microscopyl Cells have classic features (eg. small darkly stained nuclei)l Detection of free 3� ends of DNA by TUNEL assay (terminal

deoxytransferase-mediated dUTP-biotin nick end labeling)

l Gel electrophoresisl Detect DNA ladder of 180 bp intervals caused by

internucleosomal DNA cleavage

l Flow cytometryl Measure externalization of phosphatidylserine (PS) with

fluorescently labeled Annexin-Vl Measure DNA fragmentation with propidium iodide

fluorescence 15

Analysis of DNA content with a flow cytometer

Recall the fluorescence intensity of the DNA dye (amount of DNA) is measured for each cell.

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Dead Cells

Determination of Cell Death by Flow Cytometery

Annexin V binds PS on the surface of cells in the early phase of apoptosis

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Triggers of apoptosis

l Programmed cell death in which many more cells are produced than survive (e.g. development of lymphocytes)

l Toxic stimuli (viruses, chemicals, ionizing radiation)

l Extracellular signals (Fas, p75 NGF-R, TNF)

l DNA damage (p53)19

C. elegans has played a key role in our understanding of Apoptosis

ced-1 mutant(No engulfment)

ced-1/ced-3(No cells die)

1090 total cells131 die

Ced-3=no death

Ced-4=no death

Ced-9=all die

H.R Horvitz and colleagues responsible for much of this work, 2002 Nobel Prize in Medicine with Sulston and Brenner.20

C. elegans apoptosis

CED-9=Blocks apoptosisCED-4=linker molecule forms activating complex with CED-3CED-3=Protease that executes cell by chewing up proteinsEGL-1=Proapoptotic by blocking CED-9 function

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Three classes of proteins function in the apoptotic pathway-conserved in

vertebrates

Mammalian Bcl-2 can substitute for Ced-9 in c. elegans

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Death�s Methods: A protease cascade

Fig 18-22

These proteases are called caspases

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Caspases

l Caspases are Cysteine directed proteases that cleave after ASPartate residues

l Ced-3 is the C. elegans homologuel At least 14 family membersl Synthesized as proenzymes with low levels of

caspase activity (~1-2 % of active form)l Activated upon after aggregation or cleavage to

mature forml Caspases –8 and –9 are �initiator� caspasesl Caspases –3 is the �effector� caspasel Caspase activation requires a stimulus l They proteolyze cellular proteins to carry out cell death program

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The Caspase Family

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Procaspase activation

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Caspase cascade

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Two Pathways that Initiate Apoptosis

l Intrinsic/ Mitochondrial Apoptosisl Regulated by Mitochondrial Cytochrome c release

l Extrinsic/ Death Receptor Apoptosisl Activated by ligation of Death Receptorsl Fas, TNF alpha

l These pathways intersect at the effector caspases

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Activation of the Intrinsic Pathway

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Intrinsic/Mitochondrial Pathway

CARD domain

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Mitochondrial permeability

PT=Permeability transition, bursts outer membrane31

Intrinsic Pathway: Apaf-1 Induced Apoptosis

CARD domains

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Smac/Diablo and IAPs

Smac=Second mitochondrial activator of caspasesIAP=Inhibitor of Apoptosis Proteins

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Bcl-2 family membersl A very large family with 19 members identifiedl Bcl-2 (homologous to ced-9) is prototypel All have the BH3 domain (Bcl-2 Homology)

l BH-3 is the pro-apoptotic domain exposed on activation

l Act as dimers=either hetero or homodimersl Pro-apoptotic dimers (Bax) increase mitochondrial

permeabilityl Anti-apoptotic members (Bcl-2, Bcl-XL) form dimers

with pro-apoptotic members to inactivate them 34

The Bcl-2 FamilyBH domains=protein-protein interaction domains

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Intrinsic Apoptosis

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Some trophic factors prevent apoptosis by inducing inactivation of a pro-apoptotic regulator

Figure 23-5037

Two mechanisms for p53 activation of apoptosis

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Extrinsic/Death Receptor Pathway

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Death Receptors and Ligands

CD95=Fas40

TNF receptor family

Death Domains (DD)Bind DDs of other proteins (e.g. FADD)…

…Recruiting them to the plasma membrane.

Cysteine-Rich Domains (CRD)

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Fas-FasL Apoptosis

l In response to antigenic stimulation, peripheral T cells expand

l The antigen specific T cells generated must be eliminated (except for the memory cells)

l Upon repeated antigenic stimulation via the T Cell receptor: T cells upregulate Fas and FasL

l Eliminate neighboring T Cells expressing Fas42

Activation of Apoptosis by Fas Ligand

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Fas Induced Apoptosis

The Formation of the Death Initiating Signal Complex (DISC)

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Adaptor Proteins contain conserved protein interaction domains

= inhibits apoptosis

-CARD domain of Apaf-1 binds CARD domain of procaspase-9.-DED domain of FADD binds DED domain of procaspase-8.-DED domains of FLIP can bind to the DED domain of FADD and block procaspase-8 recruitment.

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Fas and the intrinsic pathway:Bid

/Bax

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Proteolytic targets of effector caspases

l Cytoskeletal regulatory proteinsl Actin

l Nuclear Laminsl Poly(ADP-ribose) polymerase (PARP)

l PARP activity depletes ATP, thus cleavage of PARP may maintain store of ATP to drive apoptosis

l DNA-fragmentation factor (DFF)

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Removal of apoptotic cell by phagocytosis

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Removal of cell corpses

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Phagocytosis: Necrosis vs. Apoptosis

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Production of anti-inflammatory cytokines and chemokinesNo up-regulation of co-stimulatory molecules

Production of pro-inflammatory cytokines and chemokinesUp-regulation of co-stimulatory molecules

Phagocytosis tags and receptors

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Two roads to activate apoptosisExtrinsic Intrinsic

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Caspase 3, 6, 7

Apoptosis

Pro-apoptotic ligand

FADDFLIP

DR5

DR4

Cell-extrinsicpathway

Procaspase 8, 10

p53p53

Caspase 9

Caspase 8, 10

p53

BAX, BAK

Mitochondria

SMAC/DIABLO

ChemotherapyRadiotherapy

DNA damage

PUMA, NOXA

APAF1

Cytochrome c

DNA damage

BID

IAP

Cell-intrinsicpathway

BCL2, BCLXL, MCL1

TNFα Can also activate Necroptosis

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Necroptosis: RIP3 Dependent programed necrosis

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Autophagy

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Two major functions:

o to eliminate damaged or superfluous proteins and organelles, commonly referred to as the protein and organelle quality control function of autophagy.

o to degrade and recycle intracellular components to sustain sustain metabolism and homeostasis in the absence of external nutrients, commonly referred to as the catabolic function of autophagy.

Autophagy, literally means ‘self-eating’.

The Autophagic Process

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Autophagy under the microscope

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Autophagic Vesicles LC3-II punctae

membrane recruitment of both PKB and its activatorPDK1 (phosphoinositide-dependent protein kinase 1),leading to the activation of PKB. PtdIns3K activity canbe opposed by PTEN, a 30-phosphoinositide phospha-tase, subsequently decreasing PKB activity, and inhibit-ing mTOR. PtdIns3K–PKB activation suppressesautophagy in mammalian cells. PKB further activatesmTORC1 through inhibiting a downstream protein com-plex, the tuberous sclerosis complex 1/2 (TSC1/TSC2).The TSC1/TSC2 heterodimer, which is a stable complex,senses the upstream inputs from various kinases, in-cluding PKB and ERK1/2 [35,36]. Phosphorylation ofTSC2 by PKB or ERK1/2 leads to the disruption of itscomplex with TSC1, and results in mTOR activation.

TSC1/TSC2 acts as the GTPase-activating protein forRheb, a small GTP-binding protein that binds to andactivates mTOR in its GTP-bound form. Ras has oppos-ing roles in autophagy regulation: it inhibits autophagy byactivating the PtdIns3K–PKB–mTORC1 pathway, andat the same time, it may induce autophagy via theRaf-1–MEK1/2–ERK1/2 pathway [37,38]. Finally, themTORC2 complex is also involved in autophagy regu-lation. Full activation of PKB requires mTORC2 [39],and inhibition of PKB, caused by mTORC2 depletion,reduces the phosphorylation of, and therefore activates,the forkhead box O (FoxO3) transcription factor, whichstimulates autophagy in muscle cells independent of theactivity of mTORC1 [40].

128 Cell regulation

Figure 2

Signaling cascades involved in the regulation of mammalian autophagy. Autophagy is regulated by a complex signaling network of various stimulatory(arrowheads) and inhibitory (bars) inputs. Activation of growth factor receptors stimulates the class I PtdIns3K complex and small GTPase Ras, whichleads to activation of the PtdIns3K–PKB–mTORC1 pathway and the Raf-1–MEK1/2–ERK1/2 pathway, respectively. PKB and ERK1/2 phosphorylateand inhibit the GTPase-activating protein complex TSC1/TSC2, leading to the stabilization of Rheb-GTPase, which, in turn, activates mTORC1,causing inhibition of autophagy. Activated ERK1/2 also stimulates autophagy. mTORC2 inhibits autophagy through the phosphorylation and activationof PKB. Metabolic stress, such as high AMP/ATP ratios resulting from energy depletion, or an increase in the cytosolic free Ca2+ concentration orcytokines, cause the AMP-activated protein kinase (AMPK) to be phosphorylated and activated by LKB1, CaMKKb, and TAK1, respectively. AMPKphosphorylates and activates TSC1/TSC2, leading to inactivation of mTORC1 and autophagy induction. Genotoxic and oncogenic stresses result innuclear p53 stabilization and activation, which stimulates autophagy through activation of AMPK or upregulation of DRAM. In contrast, cytosolic p53has an inhibitory effect on autophagy. Antiapoptotic proteins, Bcl-2 or Bcl-XL, associate with Beclin 1 and inhibit the Beclin 1-associated class IIIPtdIns3K complex, causing inhibition of autophagy. For additional details, see the main text.

Current Opinion in Cell Biology 2010, 22:124–131 www.sciencedirect.com

Signals Controlling Autophagy

Current Opinion in Cell Biology 2010, 22:124–131

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Genotoxic and oncogenic stresses result in

nuclear p53 stabilization and activation, which

stimulates autophagy through activation of

AMPK

AMPK phosphorylates and activates the GAP

complex TSC1/TSC2, leading to inactivation of

mTORC1 and autophagy induction.

Rheb is a GTPase

AMPK->mTORC1 activates autophagy

PI3K-> AKT blocks autophagy

Autophagy in Cancer

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Autophagy Can promote tumor formation or block it depending on context.

Clin Cancer Res; 21(3): 498

Questions?

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