Download APOPTOSIS: An overview

CHAP16.APOPTOSIS
INTRODUCTION
Apoptosis or programmed cell death, is carefully
coordinated collapse of cell, protein degradation,
DNA fragmentation followed by rapid
engulfment of corpses by neighbouring cells.
(Tommi, 2002)
Essential part of life for every multicellular
organism from worms to humans. (Faddy et al.,1992)
Apoptosis plays a major role from embryonic
development to senescence.
WHY SHOULD A CELL COMMIT SUICIDE?
Apoptosis is needed for proper development
Examples:
The resorption of the tadpole tail
 The formation of the fingers and toes of the fetus
 The sloughing off of the inner lining of the uterus


The formation of the proper connections between neurons in the brain
Apoptosis is needed for self defense
Examples:
Cells infected with viruses
 Cells of the immune system
 Cells with DNA damage
 Cancer cells

Apoptosis in physiologic
situations
Programmed cell death during embryogenesis
Formation of
free and
independent
digits
Development
of the brain
Development
of
reproductive
organs
WHAT MAKES A CELL DECIDE TO COMMIT SUICIDE?
Withdrawal of positive signals
examples :
 growth factors for neurons
 Interleukin-2 (IL-2)
Receipt of negative signals
examples :
 increased levels of oxidants within the cell
 damage to DNA by oxidants
 death activators :
Tumor necrosis factor alpha (TNF-)
 Lymphotoxin (TNF-β)
 Fas ligand (FasL)

CAUSES OF CELL DEATH:
Cell death by injury
-Mechanical damage
-Exposure to toxic chemicals
Cell death by suicide
-Internal signals
-External signals
HISTORY OF CELL DEATH / APOPTOSIS
RESEARCH
1800s
1908
1930-40
1948-49
1955
1964-66
1971
1977
1980-82
1989-91
Numerous observation of cell death
Mechnikov wins Nobel prize (phagocytosis)
Studies of metamorphosis
Cell death in chick limb & exploration of NGF
Beginning of studies of lysomes
Necrosis & PCD described
Term apoptosis coined
Cell death genes in C. elegans
DNA ladder observed & ced-3 identified
Apoptosis genes identified, including bcl-2,
fas/apo1 p53, ced-3 sequenced
(Richerd et.al., 2001)
NECROSIS VS. APOPTOSIS
Necrosis
•
•
•
•
Cellular swelling
Membranes are broken
ATP is depleted
Cell lyses, eliciting an
inflammatory reaction
• DNA fragmentation is
random, or smeared
• In vivo, whole areas of
the tissue are affected
Apoptosis






Cellular condensation
Membranes remain intact
Requires ATP
Cell is phagocytosed, no
tissue reaction
Ladder-like DNA
fragmentation
In vivo, individual cells
appear affected
NECROSIS VS APOPTOSIS
Wilde, 1999
STAGES OF APOPTOSIS
Induction of apoptosis related genes, signal transduction
Sherman et al., 1997
APOPTOSIS: Morphology
organelle
membrane
blebbing &
changes
reduction
cell
mitochondrial
leakage
shrinkage
nuclear
fragmentation
chromatin
condensation
Hacker., 2000
APOPTOSIS: Morphological events
cell shrinkage
organelle reduction
mitochondrial leakage
chromatin condensation
nuclear fragmentation
membrane blebbing & changes
Blebbing & Apoptotic bodies
Bleb
The control retained over the cell
membrane & cytoskeleton allows intact
pieces of the cell to separate for
recognition & phagocytosis by MFs
Apoptotic body
MF
MF
Mechanisms of Apoptosis
The fundamental events in apoptosis is the activation of enzymes called
CASPASES
Caspases
• Cysteine proteases
• Cysteinedependent
ASPartate-specific
proteASES
APOPTOSIS: PATHWAYS
“Extrinsic Pathway”
Death
Ligands
Death
Receptors
“Intrinsic Pathway”
DNA
damage
& p53
Mitochondria/
Cytochrome C
Initiator
Caspase 8
Effector
Caspase 3
Initiator
Caspase 9
PCD
INITIATION
•
Absence of stimuli - hormones, growth factors
•
Activation of receptors – TNF family
•
Heat ,radiation, chemicals
•
Genetically programmed events
PATHWAY OF APOPTOSIS

Initiation
• Intrinsic pathway
• Extrinsic Pathway
• Cleavage of DNA
Execution
Phagocyt-osis
• Engulfment of cell
Mitochondrial pathway
• Intrinsic pathway
The death receptor
pathway
• Extrinsic pathway
Mitochondrial pathway
• Intrinsic pathway
Mitochondria
BAX
BAK
BOK
BCL-Xs
BAD
BID
B IK
BIM
NIP3
BNIP3
BCL-2
BCL-XL
BCL-W
MCL1
BFL1
DIVA
NR-13
Several
viral
proteins
• Extrinsi
c
pathwa
y
The death
receptor
pathway
Bcl-2 family members
A very large family with 30 members identified and belongs to both
BH1,
BH2,BH
3
Antiapoptotic
Proapoptotic
BH1,
BH2,BH3,B
H4
Bid
Bim
Bik
Bad
Bmf
Hrk
Noxa
BH
Puma
3
Blk
BNIP3
Spike
MAJOR PLAYERS IN
APOPTOSIS
•
•
•
•
Caspases
Adaptor proteins
TNF & TNFR family
Bcl-2 family
LIGAND-INDUCED CELL DEATH
Ligand
Receptor
FasL
TNF
TRAIL
Fas (CD95)
TNF-R
DR4 (Trail-R)
LIGAND-INDUCED CELL DEATH
“The death receptors”
FasL
Ligand-induced trimerization
Trail
TNF
Death Domains
Death Effectors
Induced proximity
of Caspase 8
Activation of
Caspase 8
APOPTOSIS: Signaling & Control pathways I
Externally driven
Apoptotic signals
p53
Internally Cytochrome C
driven
Externally driven
Activators of
initiator enzymes
Initiator caspases
mitochondrion
6, 8, 9,12
Execution caspases
2, 3, 7
Apoptosis events
Activation
APOPTOSIS: Signaling & Control pathways II
Externally driven
Apoptotic signals
p53
Internally Cytochrome C
Bcl2
driven
Externally driven
Inhibitors
Activators of
initiator enzymes
Initiator caspases
6, 8, 9,12
External Survival
Internal factors
Execution caspases
2, 3, 7
Apoptosis events
Inhibitors of
apoptosis
Inhibition
The mitochondrial pathway
DNA
Fas
damage
Casp8
p53
Growth factor
receptors
PI3K
Bid
Bid
Bax
Bid
Bax
Akt
casp3
BAD
Bcl2
casp9
Apaf1
ATP
Cyt.C
IAPs
casp3
Smac/
DIABLO
H2O2
AIF
Pollack etal., 2001
Importance of Apoptosis
• Important in normal physiology / development
– Development:
– Immune system maturation
– Morphogenesis
– Neural development
– Adult:
– Immune privilege
– DNA Damage
– Wound repair.
INACTIVATING P53 PATHWAY IN
CANCER
P53 INACTIVATION IN HUMAN CANCER
•
p53 mutations can be found in 50% of human cancers, but their penetrance is
highly heterogeneous, as reflected by the diverse remaining transactivation
activity that ranges from O to 100%.
•
Various DNA viruses, such as SV40, HPV or adenoviruses, encode proteins
that target p53 protein.
•
mdm2 accumulation is found in numerous cancers, such as sarcoma or
breast carcinomas.
•
PTEN, a p53 regulated gene, is mutated in various types of cancer including
glioblastoma.
•
Although no mutation of AKT has been found in human cancer, constitutive
activation of its kinase activity has been observed via deregulation of the
upstream pathway.
•
Mutations in various pathways upstream of p53 (ATM, p19ARF or Hcdk2
gene) can be observed in various types of cancer.
Immune system maturation:
Positive selection of thymocytes in the
thymus. Thymic selection involves
thymic stromal cells (epithelial cells,
dendritic cells, and macrophages), and
results in mature T cells that are both
self-MHC restricted and self-tolerant.
Immune system maturation:
Negative selection of thymocytes in the
thymus. Thymic selection involves
thymic stromal cells (epithelial cells,
dendritic cells, and macrophages), and
results in mature T cells that are both
self-MHC restricted and self-tolerant.
Morphogenesis:

Molecular basis:
Morphogens
 Transcription factors
 Cell adhesion molecules


Cellular basis:
Cell-cell adhesion
 Cell contractility
 Extracellular matrix
 Apoptosis

Immune Privilege:

Fas-ligand (FasL; also called CD95L or Apo-1L)

required for tissues to display a privileged status

FasL functions to induce apoptotic cell death in most
cells that express its receptor, Fas.

Fas-bearing cells include cells of the immune
system

Tissues that naturally express FasL kill
infiltrating lymphocytes and inflammatory cells.
DNA Damage
Ionizing Radiation,
Carcinogens & Mutagens
Role of apoptosis
In maintaining
Integrity of genomic
DNA in normal cells
DNA Damage
p53 activated and binds to DNA
Transcriptional Up-Regulation
of Target Genes
p21
GADD45
BAX
(CDK inhibitor)
(DNA repair)
(apoptosis gene)
G1 Arrest
Successful repair
Repair fails
APOPTOSIS
Disruption of apoptosis
 Two

major ways:
Inappropriate activation of the
apoptotic process
Immune defect in AIDS
 Neurodegenerative diseases.


Inadequate apoptosis
Cancer
 Chronic inflammatory conditions
 Autoimmune diseases.

Immune defect in AIDS




Profound reduction in the population size of CD4
+ T helper cells
Caused by excessive apoptosis
Process includes transfer of regulatory viral gene
products (such as HIV-1 Tat) from HIV infected
cells to bystander T cells
Renders them susceptible to T cell receptorinduced, CD95-mediated apoptosis.
Neurodegenerative Disease

Apoptosis triggered by
amyloid β
 neurotoxic abnormal protein structures or aggregates


In adult neurodegenerative diseases including
Alzheimer's
 Huntington's chorea
 Parkinson's disease,
 Amyotrophic lateral sclerosis


Amyloid β can exert neurotoxic effects by
generation of intracellular oxidative stress
 increases in calcium ions


Both of these can trigger apoptosis in susceptible
cell types.
The bcl-2 family
N
BH4
BH3
BH1
Receptor domain
Ligand
Pore
domain
formation
phosphorylation
Raf-1
calcineurin
BH2
TM
C
Membrane
anchor
Group I
Bcl-2
Group II
bax
Group III
Bad
bid
bik
Back
P53 & Apoptosis
p53 first arrests cell growth between G1  S
This allows for DNA repair during delay
If the damage is too extensive then p53
induces gene activation leading to
apoptosis (programmed cell death)
BACK
Cells are balanced between life and death
DAMAGE
Physiological death signals
sclero dinesh
DEATH SIGNAL
ANTIAPOPTOTIC
PROTEINS
(dozens!)
PROAPOPTOTIC
PROTEINS
(dozens!)
DEATH