APOPTOSIS

• Formed by Greek prefix apo- (from) and root ptosis (falling)

• “leaves falling from a tree……”
• the differentiation of fingers and toes in a developing human
embryo occurs because cells between the fingers apoptose;
the result is that the digits are separate.
• Between 50 and 70 billion cells die each day due to apoptosis
in the average human adult
• For an average child between the ages of 8 and 14,
approximately 20 billion to 30 billion cells die a day
• Cells are born, live for a given period of time and then die-
• Physiological cell death
• Cell suicide
• Cell deletion
• Programmed cell death
AN APOPTOTIC CELL IN CULTURE

Collins JA, et al. 1997

 THE APOPTOTIC PATHWAY

Triggers Modulators Effectors Substrates DEATH

. Growth factor . FADD . Caspases . Many cellular

Deprivation . TRADD proteins
. Hypoxia . FLIP . DNA
. Loss of adhesion . Bcl-2 family
. Death receptors . Cytochrome c
. Radiation . p53
. Chemotherapy . Mdm2
 Apoptosis
Apoptosis is a cell death process which occurs during development and aging
of animals.
Death by injury
The cells swell and cell contents leak out, leading to inflammation of
surrounding tissues
Death by suicide
The process is often called programmed cell death or PCD.

German scientist Carl Vogt was first to describe the principle of apoptosis in 1842.

In 1885, anatomist Walther Flemming delivered a more precise description of the

process of programmed cell death.

The 2002 Nobel Prize in Medicine was awarded to Sydney Brenner, Horvitz and John E.
Sulston for their work regarding apoptosis
 Etiology of cell death

Necrosis:
• The sum of the morphologic changes that follow cell death in a living
tissue or organ
Apoptosis:
• A physiological process that includes specific suicide signals leading to
cell death
 Forms of cell death
"Classic"
Necrosis Apoptosis Mitotic catastrophe

Passive Active Passive

Pathological Physiological or Pathological

pathological

Swelling, lysis Condensation, Swelling, lysis

cross-linking

Dissipates Phagocytosed Dissipates

Inflammation No inflammation Inflammation

Externally induced Internally or Internally induced

externally induced
 APOPTOSIS
In development, apoptosis removes unwanted cells; for
example in separating the digits of the hand, culling
excess neurons, eliminating inappropriately sensitive
lymphocytes
In mature tissues that undergo renewal, e.g., blood cells,
some epithelia, the outdated cells deliberately destroy
themselves, if they can
Some defensive cells die in the course of protecting the
body, e.g. neutrophils, or in developing maximum
affinity for attacking an invader, e.g. B lymphocytes
Infected cells may be removed by apoptosis
Most tumor cells grow; some become apoptotic
Apoptosis is used in running down unused or unwanted
female reproductive tissue as it cycles, e.g., uterine
endometrium, corpus luteum, breast gland epithelium
 APOPTOSIS: Contexts II
In development, apoptosis removes unwanted cells,
more examples: after matrix synthesis in the growth
plate, in old bone awaiting resorption,
Some defensive cells die in the course of protecting the
body: lymphocytes can kill each other, thus limiting
immune reactions, & avoiding autoimmunity
Apoptosis is part of running down unused or unwanted
female reproductive tissue as it cycles, e.g., uterine
endometrium, corpus luteum, breast secretory epithelium

In ovarian late atretic (degenerating) follicles,

granulosa cells display hallmarks of apoptosis,
e.g., fragmented nuclei, shrinkage, as they shed
into the lumen
 APOPTOSIS: Contexts III
In these contexts, two kind of situation emerge: tidy
coordinated loss (and sometimes replacement) of cells;
and the messy battlefields of defense against infection,
reaction to tumors, & wound repair

The macrophages go about their business of cell

disposal very discreetly, without inflammation

This is not death with the headlights on and the traffic

stopped; rather it is akin to the night-time anatomical
grave-robbing of old, except that for apoptosis the body
may be dismembered, but is not quite dead
 APOPTOSIS: Contexts IV
Another context is all the other paths open to a cell,
aside from apoptosis
Histology is first presented mostly as a described steady
state - this is what the cells look like, and what they are
doing as their normal function
However, the cells have many options, and are held by
numerous controlling systems in this ‘steady state’

The options open to a cell can be categorized as: keep

going/holding steady; advance; or retreat; as depicted
on the next figures

Programmed cell death is the last resort of retreat

[Programmed cell death is not quite synonymous with apoptosis,
since the latter term is restricted by some biologists to
programmed cell death dependent on caspase enzymes]
 APOPTOSIS: Contexts VI - Cell options

apoptosis keep working

fuse
stress responses

hypertrophy
shrink

enlarge &
divide
de-differentiate transdifferentiate
senescence
RETREAT OR ADVANCE
 APOPTOSIS: Contexts VII
However, the cells have many options, and are held by
many controlling systems in this ‘steady state’

These categories - keep going; advance; or retreat - are

mutually exclusive, so that control systems have to be
interlocked, with inhibition of contrary activities, while
stimulating a particular sequence and path
This integrated control requires a host of chemical
agents and factors, and has to take into account the
many circumstances in which a cell finds itself

Few of these factors that regulate apoptosis: p53, Bcl-2

family, cytochrome c, TNF-, Fas ligand & receptor,
caspases, endonucleases
Apoptosis displays apoptosis
features of the cell states
that often precede it

stress responses

Apoptosis often occurs

because the less drastic
state of retreat cannot be shrink
maintained

de-differentiate
RETREAT OR
 APOPTOSIS: Morphology

organelle
reduction
membrane
blebbing &
changes
cell
mitochondrial
leakage shrinkage

nuclear chromatin
fragmentation condensation
 APOPTOSIS: Morphological events
cell shrinkage
organelle reduction
mitochondrial leakage
chromatin condensation
nuclear fragmentation
membrane blebbing & changes
 APOPTOSIS: Morphological events
organelle reduction
sounds a rather coy term, but is intended to indicate
that the organelles change and become non-functional,
in ways often short of fast, outright destruction; e.g.,

Chiu R et al. A caspase cleavage fragment of p115

induces fragmentation of the Golgi apparatus and
apoptosis. J Cell Biol 2002;159:637-648
 Minimum for cell death
The cell depends on the nucleus, so take this
out of commission with enzymes for
chromatin condensation
nuclear fragmentation

Call in the macrophages with a surface marker

membrane changes

However, for orderliness, and to help the

macrophages, other changes occur,
mitochondrial leakage which convert the cell to
wrapped, smaller,
organelle reduction partially digested pieces
cell shrinkage
Wrapped - cell membrane
membrane blebbing integrity is kept
Blebbing & Apoptotic bodies
The control retained over the cell
Bleb membrane & cytoskeleton allows intact
pieces of the cell to separate for
recognition & phagocytosis by Ms

Apoptotic body

M M
 Macrophage recognition of the apoptotic cell
Call in the macrophages with a surface marker

membrane changes

Apoptotic body

M
Plasma membrane changes:

Phosphatidylserine is exposed externally

Membrane proteins lose their normally
M the membrane
asymmetric distribution across
 Time for some chemical agents
The cell depends on the nucleus, so take this
out of commission with enzymes for
chromatin condensation
nuclear fragmentation ENDONUCLEASES
Call in the macrophages with a surface marker
membrane CD14, & ligands for
changes, e.g. macrophageCD44

However, for orderliness, and to help the

macrophages, other changes occur,
mitochondrial leakage Cytochrome c
organelle reduction
cell shrinkage CASPASE proteases

membrane blebbing
CASPASE cysteinyl-aspartate-specific proteinase
 APOPTOSIS: Signaling & execution
Fas ligand Tumor Necrosis Factor- Apoptotic signals

Initiator caspases

Cytochrome c

Execution caspases

Apoptosis events
 Apoptosis vs. Necrosis
• Necrosis
– Swelling of cell
– Disassembly of organelles
– Membrane integrity
compromised
– Release of cell contents
– Often lead to inflammatory
response
• Apoptosis
– Shrinkage of cell
– Preservation of membrane
integrity
– Chromatin condensation
– Phagocytosed by other cells
– No inflammatory response
 Apoptosis and Programmed Cell
Death (PCD)
• These two term are usually used
interchangeably
• Apoptosis emphasizes more about the
morphology (to be mentioned below)
• PCD emphasizes more about the
orchestrated nature
• PCD is a broader term than apoptosis (PCD
in plant, autophagic cell death, etc.)
 So what is apoptosis?
• Regulated cell death
• Intrinsic mechanism
• Unique cell morphology
• Crucial in development and diseases
 Morphology of Apoptosis
• Cell shrinkage (blebbing)
• Dark Nucleus (due to disintegration of
chromatin)
• Apoptotic body
 Morphology of Apoptosis
• Cell shrinkage
(blebbing)
• Dark Nucleus (due to
disintegration of
chromatin)
• Apoptotic body
 Morphology of Apoptosis
• Cell shrinkage
(blebbing)
• Dark Nucleus (due to
disintegration of
chromatin)
• Apoptotic body
 Morphology of Apoptosis
• Cell shrinkage
(blebbing)
• Dark Nucleus (due to
disintegration of
chromatin)
• Apoptotic body
How Apoptosis was Discovered?
• First discovered in 1970’s during research
of Caenorhabditis elegans development
• The discoverer, Dr. Horvitz, won 2002
Nobel Prize in Physiology or Medicine
• Has since been found in many higher
animals (including human, of course).
 Caenorhabditis elegans
• A nematode (app.
1mm long)
• A model animal in
molecular and
development biology
research
• Genome determined
 A perfect model for development
biology
• Short life cycle (3 weeks)
• Existence of both allogamy and autogamy.
• Whole development is determined to the
single cell level
– Every C. elegans has exactly 1090 somatic cells
– Each cell’s fate is invariant in all individuals
 Apoptosis in C. elegans
• Of the 1090 cells C. elegans has, exactly 131 will
undergo apoptosis, making the adult nematode has
only 959 cells.
• Mutations affecting the death of these cells are
identified. These mutants are viable
• ced-3 and ced-4 were first identified as pro-
apoptotic genes (killer genes)
• ced-9 later was found to be anti-apoptotic genes
 Not just in those worms!
• The gene product of ced-3 was found to be
homologous to a mammalian protein ICE
(later dubbed as caspase 1)
• Caspases were found in mammals, as well
as other animals such as zebrafish and fruit
fly
• Now apoptosis is generally believed to be a
ubiquitous phenomenon among metazoa
 Apoptosis in Development
• An efficient way to get rid of cells no
longer needed
• Examples:
– Digit development
– Elongation of long bone
– Amphibian metamorphosis
 Apoptosis in Development
• Digit development
• Elongation of long
bone
• Amphibian
metamorphosis
 Apoptosis in Development
• Digit development
• Elongation of long
bone
• Amphibian
metamorphosis
 Apoptosis in Development
• Digit development
• Elongation of long
bone
• Amphibian
metamorphosis
STAGES OF CLASSIC APOPTOSIS
Healthy cell

DEATH SIGNAL (extrinsic or intrinsic)

Commitment to die (reversible)

EXECUTION (irreversible)

Dead cell (condensed, crosslinked)

ENGULFMENT (macrophages, neighboring cells)

DEGRADATION
 STAGES OF CLASSIC APOPTOSIS

Genetically controlled: Caenorhabditis elegans

soil nematode (worm)

ces2 ces1 ced9 ced3,4

Healthy cell Committed cell Dead cell

BCL2 Caspases
(proteases)

C. elegans genes == mammalian genes

Cells are balanced between life and death
DAMAGE Physiological death signals

DEATH SIGNAL

PROAPOPTOTIC ANTIAPOPTOTIC
PROTEINS PROTEINS
(dozens!) (dozens!)

DEATH
 APOPTOSIS: important in embryogenesis

Morphogenesis (eliminates excess cells):

Selection (eliminates non-functional cells):

 APOPTOSIS: important in embryogenesis

Immunity (eliminates dangerous cells):

Self antigen
recognizing cell

Organ size (eliminates excess cells):

 APOPTOSIS: important in adults
Tissue remodeling (eliminates cells no longer needed):

Apoptosis
Virgin mammary gland Late pregnancy, lactation Involution
(non-pregnant, non-lactating)

- Testosterone
Apoptosis

Prostate gland
 APOPTOSIS: important in adults
Tissue remodeling (eliminates cells no longer needed):

Apoptosis

Resting lymphocytes + antigen (e.g. infection) - antigen (e.g. recovery)

Steroid immunosuppressants: kill

lymphocytes by apoptosis

Lymphocytes poised to die by apoptosis

 APOPTOSIS: important in adults
Maintains organ size and function:

Apoptosis
X
+ cell division

Cells lost by apoptosis are replaced by cell division

(remember limited replicative potential of normal cells

restricts how many times this can occur before
tissue renewal declines)
 APOPTOSIS: control
Receptor pathway (physiological):
FAS ligand TNF

Death receptors:
(FAS, TNF-R, etc) Death
domains

Adaptor proteins

Pro-caspase 8 (inactive) Caspase 8 (active)

Pro-execution caspase (inactive)

Execution caspase (active)

MITOCHONDRIA Death
 APOPTOSIS: control
Intrinsic pathway (damage):
Mitochondria

BAX Cytochrome c release BCL-2

BAK BCL-XL
BOK BCL-W
BCL-Xs Pro-caspase 9 cleavage MCL1
BAD BFL1
BID DIVA
B IK NR-13
BIM Pro-execution caspase (3) cleavage Several
NIP3 viral
BNIP3 proteins

Caspase (3) cleavage of cellular proteins,

nuclease activation,
etc.

Death
Apoptosis and Proliferation: Two
faces of a coin
Diseases Related with Apoptosis
• Decreased rate of apoptosis let cells that usually
die survive. Why is this bad?
• When cells have DNA damage beyond repair, they
normally undergo apoptosis. If they somehow
escape apoptosis (mostly related to p53 mutation),
cancer might follow
• Some viruses can hijack the host transcriptional
system to produce anti-apoptotic proteins to
prevent their host from apoptosis
Diseases Related with Apoptosis
• Too much apoptosis causes atrophy
• For aging people, their neurons may
undergo apoptosis, causing
neurodegenerative diseases (Alzheimer's,
Parkinson’s)
• Alcohol-induced liver diseases
• Myocyte degeneration
Caspases: Executioner of Apoptosis
• A family of Cysteine-aspartic-acid-
proteinases (14 is known)
• With cysteine in active site
• Cleave after aspartic acid
• Normally in inactivated form (pro-enzyme)
• Can be activated via cleavage after aspartic
acid (hint: self-activation)
• Use positive feedback to form a cascade
 Initiator and Effector Caspases
• Initiator caspases (2, 9, 8, 10):
– Activated by intrinsic or extrinsic stimuli
– Cleave and activated effector caspases:
• Effector caspases (3, 6, 7):
– Activated by initiator caspases
– Cleave and activate themselves and carry out caspase
cascade
– Cleave many other proteins to fulfill the whole
apoptosis phenomena
 What lead to caspases?
• Two general pathways involved
– Death-receptor
– Mitochondrial
• Different initiator caspases are used
• Both pathways converge in caspase 3, the
main effector caspase
 Death-receptor Pathway
• Triggered by extrinsic ligand (“order of seppuku” ?)
• A family of membrane receptors, generally called death-
receptors, regulates this pathway. e.g. TNF I, CD95
• These receptors recruit procaspase-8 via an adaptor protein
(FADD)
• Caspase-8 activation by proximity induction
• Effector caspases…
 Mitochondria Pathway
• Triggered by intrinsic signals, normally when the cell is damaged
beyond repair
• Activation of some members of an important protein family (bcl-2
family)
• Mitochondria release cytochrome C
• Formation of apoptosome (cytochrome C, Apaf-1, caspase-9)
• Caspase-9 is activated
Early in apoptosis, mitochondria are triggered by multiple
stimuli to release proteins that induce apoptosis. These
include: oxidants, bax (a pro-apoptotic protein that targets
mitochondrial membranes), Ca2+ overload, active caspases,
and perhaps ceramide
The following caspase-activating proteins are then
released from the intermembrane space:
1. Cytochrome c (SMAC)
2. AIF (apoptosis-inducing factor) OMNI
3. And procaspases like procaspase-3 and caspase-2
Cytochrome c binds with Apaf-1, which then associates with
procaspase- 9. This triggers caspase-9 activation. The complex of
cytochrome c-Apaf- 1-caspase-9 then activates caspase-3
proteolytically.
AIF also processes procaspase-3 to initiate caspase-3 activation.
This cascade by caspases (cysteine proteases that cleave substrates
at aspartic acid residues) culminates in apoptosis.
There are two general mechanisms:
1. The outer mitochondrial membrane ruptures due to
expansion of the matrix space and organellar swelling.
2. This releases cytochrome c, AIF, etc.
How are cytochrome c and other caspase-activating pro
released from mitochondria?
In this scenario, the mitochondrial inner membrane potential
drops, indicating the openings of channels known at
permeability transition pores. These pores are composed
of both inner and outer membrane proteins.
When the pores open, water and solutes enter the matrix,
causing matrix swelling and outer membrane disruption.
2. The other mechanism also involves the opening of
channels. But, in contrast, these permeability transition
pores open only in the outer membrane and do not result
in organellar swelling.
These transition pores allow cytochrome c and other
proteins to move from the intermembrance space into the
cytosol.
•Permeability of the membranes appear to be
enhanced by calcium, pro-oxidants, and several
apoptosis-related proteases (caspases)
•Bcl-2 and Bcl-2-like proteins increase resistance to
pore opening
 Bcl-2 family
• A protein family with its membranes related
tightly with apoptosis
• At least 14 members
• 3 Groups
 Bcl-2 family
• Group 1 contains BH4 domain,
usually anti-apoptotic
• Group 2 and 3 usually pro-
apoptotic
• Yin and yang
• Cell survival/apoptosis depends
on balance of these two anti and
pro-apoptotic proteins
Possible Mechanism of Bcl-2 Family
Protein Function
• The pro-apoptotic members of Bcl-2 family for a
pore on mitochondrial membrane through which
cytochrome C can be released
• They can also directly activate caspase via adaptor
molecules
• They can modulate mitochondrial membrane to
control the chemical and energy balance across the
membrane
• The anti-apoptotic members bind to the pro-
apoptotic members to form a heterodimer, so as to
disrupt the aforementioned functions
 Ways to detect Apoptosis
• Morphological evidence (mentioned above)
• Phosphatidylserine translocation
• Caspase activation
• DNA fragmentation
Phosphatidylserine Translocation
• Phosphatidylserine (PS) is a kind of
phospholipid that constitutes the
plasma membrane
• In normal cells, PS always resides in
the cytosol side of the plasma
membrane
• In apoptotic cells, PS quickly
translocates to the exterior side of
plasma membrane, which tags the
apoptotic cells for engulfment by
other cells (the “eat me” signal)
• The translocated PS can be detected
by annexin-V binding
Phosphatidylserine Translocation
(Detected by Flow Cytometry)
 DNA Fragmentation
• In normal cells, DNA are
wired around protein
spindle called histones
• DNA and histones form
units called nucleosomes
• In apoptotic cells, cleaved
by DNase, nucleosomes
are cut loose, like beads
come off a string
 DNA Fragmentation
• DNA of apoptotic cells
subject to electrophoresis
• The DNA in nucleosomes
cut loose have lower MW
than intact DNA, thus
move faster in
electrophoresis
• Result in a “ladder” in the
gel
 Calbiochem, Inc

CELL SURFACE DEATH RECEPTORS

 APOPTOTIC SIGNALING PATHWAYS
Growth Factor Withdrawal
? Irradiation
Loss of Matrix Contact
Glucocorticoids
Bax/Bak
FADD
Fas
Bcl-2
Bcl-X Cytochrome c
m
tBid
Procaspase 8 Smac Apaf-1
IAP’s
Procaspase 9
C-FLIP Bid
Caspase 9
Caspase 8

iCAD Procaspase 3
Procaspase 3
CAD
Caspase 3 Caspase 3
DNA Fragmentation
EXTRINSIC PATHWAY INTRINSIC PATHWAY
 CASPASES
Caspase-1 (ICE)
Caspase-2 (ICH-1, Nedd-2)
Caspase-3 (CPP32, Apopain, Yama)
Caspase-4 (ICH-2, TX, ICEreıı)
Caspase-5 (ICErelııı, TY)
Caspase-6 (Mch2)
Caspase-7 (ICE-LAP3, Mch3, CMH-1)
Caspase-8 (FLICE, Mch5, MACH)
Caspace-9 (Mch6, ICE-LAP6)
Caspase-10 (Mch4)
SUBSTRATES for CASPASES
... PARP
... DNA-PK
... pRb
... Lamins
... NuMA
... Fodrin
... -Aktin
... Mdm2
... Cyclin A2
... Presenilin
... Others
WHERE can APOPTOSIS be
ENCOUNTERED ?

... Growth of Embrio

... Tissue Homeostasis
... Immunology
... Chronic viral diseases
... Neurodegenerative diseases
... Reperfusion injury
... Insuline-dependent Diabetes
... Atheroschlerosis
... Miyokard Infarction
... AIDS
... Development and Treatment of Malignancies
 Significance of apoptosis in cancer
1) Tumor induction
• Apoptosis is an important regulator of homeostasis--
elimination of cells with damaged genome or growth
in marginal (hypoxia)
• Apoptosis ensures the integrity of tissues
2) Viral transformation (adenoviral model) uses both
proliferation stimulus (E1A) and anti-apoptotic (E1B)
mechanisms.
3) Anti-neoplastic agents can cause an apoptotic tumor
cell death
 Moduators of Apoptosis--bcl-2 Family
• Bcl (B cell lymphoma)-2 Bcl-2 Family
cloned from Dimerization
chromosomal Anti-apoptosis Pore Formation
translocation Bcl-2 BH4 BH3 BH1 BH2 MA

Bcl-xL
• Protect from death due to
growth factor withdrawal CED-9 BH4 BH1 BH2 MA

• functional homologue of Pro-apoptosis

ced-9 Bax BH3 BH1 BH2 MA

• Pro- and anti-apoptotic

Bik
members BH3 MA

Bad BH3
Bid
EGL-1
 Effectors of Apoptosis--Caspases

• Proteases synthesized
CASPASES
as proenzyme (initiator
vs effector) Pro
(DD’s)
Large Small
(20 kd) (10 kd)
• Cysteine in active site
• Aspartate key to DX DX

recognition sequence
(iCAD)
• ced-3 prototypical
member
Active Caspase
 Caspase Substrates
Category Substrate Caspase Effect Proposed Role in Apoptosis

Structural Nuclear Lamins 6 Degraded Disassembly of nuclear matrix

-Spectrin 3 Degraded Disassemble cytoskeletal-membrane

contacts
FAK 7,6 Inactivated Disassemble focal adhesions

Gelsolin 3 Activated Disassemble actin filaments, blebbing,

DNA cleavage
Actin 3,1 Degraded Disassemble actin filaments

-catenin 3 Degraded Disassemble cell-cell contacts

NuMA 3,4,6,7 Degraded Disassemble chromatin/matrix contacts

Vimentin 3 Degraded Disassemble intermediate filaments

Signaling MEKK1 3 Activated Activate SAPK

Stat1 ? Inactivated Inhibit IFN signaling

PKC , 3 Activated Nuclear condensation/fragmentation

D4-GDI 1,3 Inactivated Deregulate Rho GTPase

Akt-1 ? Inactivated Inhibit survival pathway

NF-B p50, 65 3 Inactivated Inhibit survival gene induction

Cell cycle DNA replication 3 Inactivated Inhibit DNA replication

complex C
mdm2 3 Altered Inhibit p53

Rb 3 Inactivated Release E2F-1

DNA repair PARP 3 Inactivated DNA nicks not recognized

DNA-PK 3 Inactivated Inhibit repair of strand breaks

Other IL-1 1 Activated Inflammation

IL-16 3 Activated Inflammation

IL-18 1 Activated Inflammation, induce IFN-

ICAD/DFF-45 3 Inactivated Allow translocation of CAD into

nucleus, DNA fragmentation
Bcl-2, Bcl-X L 3 Altered Functions like BAX

Bid 8 Activated Translocates to mitochondria

Nedd4 1,3,6,7 Regulate ubiquitination

 Inhibitors of Apoptosis--IAP’s

• Bind procaspases to prevent activation

– initial description of bacculovirus product (p35)
– contain metal-binding (~80aa) BIR repeats
– number and sequence of BIR’s determine specificity of procaspase inhibition
– many contain RING fingers

• Survivin
– expressed in many fetal tissues
– expressed in many colon cancers

• IAP modulators
– inhibitors of inhibitors
– Smac/Diablo
 Pathways of Apoptosis--Mitochondria

• Cytochrome c released
• Cytochrome c binds MITOCHONDRIA

Apaf-1 (ced-4
homologue)
CASPASE 9
• Apaf-1/cytochrome c
complex binds
caspase 9 (ced-3 CASPASE 3
OTHERS
homologue) and
causes autoactivation

DEATH
 TNF Receptor Family Signaling

1 TNF
TNFR R2

FADD
Fas
TRADD
TRAF2

Procaspase 8 TRAF1

Caspase 8 NFkB Activation and

Gene Induction (eg.
SOD, IAP’s)
Procaspase 3
Anti-Apoptotic
Caspase 3

Pro-Apoptotic
Anoikis--Death by loss of matrix contact

IGF-1 Adhesion--ILK, FAK, Shc

PI-3K
PTEN

Akt
Bad, Bim, ProC9

GSK-3
Apotosis
Rb-E2F CyclinD1

Based on Yu, et.al. Mol. Cell. Biol.

21:3325.
G1S
 p53-Mediated Apoptosis

TA PP DB T CTD

H2 N COOH

TA Transactivation domain recruits proteins involved in transcription--eg

TATA box binding proteins and associated factors--also binding site
for oncogene Mdm2 which targets p53 for ubiquitination (p19 ARF
inhibits)
PP Polyproline domain critical for the induction of apoptosis and
transcriptional repression, but not activation

DB Sequence-specific DNA binding domain region of most mutations in

tumors

T Tetramerization domain important for oligomerization and DNA

binding

CTD COOH-termainal domain that is a negative regulator of the

transactivating domain
 Cell cycle and apoptosis
Cyclin B
• p53 bax and bcl-2 bax
cdc2 M G0
• overexpression of p53 Cyclin D
Rb
E2F1 causes Cyclin A
E2F1

G2 G1
apoptosis cdc2 Cyclin E
cdk4/6
• p21 binds caspase 3 R P
P
cdk2
and is a substrate Cyclin A S Rb
P P
E2F1
p21
p27
p15
p16

for caspase 3 cdk2

E2F1
Inactive p53 TGF
Adapted from Lundberg and Weinberg Eur. J. Cancer 35:531
 Anti-neoplastic drugs and apoptosis

• Caspase 9 or Apaf-1 deficient cells are resistant to many forms of anti-neoplastic

therapy (irradiation, etoposide, doxorubicin, cis-platin)
– These agents work primarily through p53, but there are poorly understood
p53-independent pathways as well
– Silencing mutations in p53 are one mechanism of drug resistance
– p53 also appears to be the target of numerous oncogenes (E1A, c-myc) and
p53 loss replaces E1B function in adenoviral transformation
• 5’-Fluoruracil appears to work through the Fas pathway in some tumors
– p53 and reactive oxygen can induce FasL and Fas expression
– Fas pathway is minor in most tumors, but neuroblastomas have silenced the
caspase 8 gene
– FADD or caspase 8 deficient fibroblasts are resistant to FasL and TNF, but
sensitive to doxorubicin and etoposide
• Akt pathway is important in tumor survival
– PTEN inactivation is correlated with poor prognosis in a number of tumor
types
 Induction of apoptosis in tumors by cytolytic
immune effectors
• Immune cells can attack tumors through the
extrinsic pathway
– TNF
– FasL
• The granule-exocytosis pathway is more
important in tumor control
– CD8 effectors
– Natural Killer (NK) Cells
– Granzymes
• Serine proteases stored in intracellular
granules
• Granzyme B is an aspase (not a caspase) that
can bypass initiator caspases to activate
effector caspases or their substrates (eg. iCAD)
• Other granzymes with different specificities--
substrates unknown
• Entry into target cells requires perforin
 APOPTOTIC SIGNALING PATHWAYS
Growth Factor Withdrawal
? Irradiation
Loss of Matrix Contact
Glucocorticoids
Bax/Bak
FADD
Fas
Bcl-2
Bcl-X Cytochrome c
m
tBid
Procaspase 8 Apaf-1

Procaspase 9
Bid
Caspase 9
Caspase 8 Granzyme B
iCAD Procaspase 3
Procaspase 3
CAD
Caspase 3 Caspase 3
DNA Fragmentation
EXTRINSIC PATHWAY INTRINSIC PATHWAY
 APOPTOSIS: control

Physiological Intrinsic
receptor pathway damage pathway

MITOCHONDRIAL SIGNALS

Caspase cleavage cascade

Orderly cleavage of proteins and DNA

CROSSLINKING OF CELL CORPSES; ENGULFMENT

(no inflammation)
APOPTOSIS: Role in Disease

TOO MUCH: Tissue atrophy

Neurodegeneration
Thin skin
etc

TOO LITTLE: Hyperplasia

Cancer
Athersclerosis
etc
 APOPTOSIS: Role in Disease
Neurodegeneration

Neurons are post-mitotic (cannot replace themselves;

neuronal stem cell replacement is inefficient)

Neuronal death caused by loss of proper connections,

loss of proper growth factors (e.g. NGF), and/or
damage (especially oxidative damage)

Neuronal dysfunction or damage results in loss of synapses

or loss of cell bodies
(synaptosis, can be reversible; apopsosis, irreversible)

PARKINSON'S DISEASE
ALZHEIMER'S DISEASE
HUNTINGTON'S DISEASE etc.
 APOPTOSIS: Role in Disease
Cancer

Apoptosis eliminates damaged cells

(damage => mutations => cancer

Tumor suppressor p53 controls senescence

and apoptosis responses to damage

Most cancer cells are defective in apoptotic response

(damaged, mutant cells survive)

High levels of anti-apoptotic proteins

or
Low levels of pro-apoptotic proteins
===> CANCER
 APOPTOSIS: Role in Disease
AGING

Aging --> both too much and too little apoptosis

(evidence for both)

Too much (accumulated oxidative damage?)

---> tissue degeneration

Too little (defective sensors, signals?

---> dysfunctional cells accumulate
hyperplasia (precancerous lesions)
OPTIMAL FUNCTION (HEALTH)

APOPTOSIS

AGING

APOPTOSIS
Neurodegeneration, cancer, …..
 Summary
• Apoptosis is a highly-regulated process that
results in an efficient removal unwanted cells. It is
greatly involved in development and prevention of
oncogenesis
• Apoptosis is a complex process that involves
interactions between many factors. The core
components of apoptosis are the caspases
• Apoptosis is very important for homeostasis of the
organism. Abnormal apoptotic rate may cause
severe diseases