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Apoptosis
Book reading club: 9th February 2007
Apoptosis: the functions
Key process in:
• Embryonic development
• Normal cellular homeostasis
Apoptosis in normal physiology:
• Intestinal epithelial cells every 4-5 days are substituted by new ones and
this process involves apoptosis
• In erythropoiesis precursors of red cells are eliminated by apoptosis. When
the levels of red cells lower there’s increase in EPO which inhibits the
apoptosis of these precursors
• Regression of the cells of mammary gland after weaning of offspring. More
or less the 90% of epithelial cells accumulated during pregnancy in the
mammary gland undergo apoptosis. This phenomenon is called involution.
Key experiments by Horovitz in ’90s
Studies on C. elegans development
• Larva of C. elegans were put on slides and single cells of the living animal
were observed as they migrated, divided and died.
• They observed that in addition to the 959 cells generated during worm
development and found in the adult, another 131 cells generated always
during the development were not present in the adult (because they
underwent programmed cell death).
• Next step was the identification of the genes responsible for the death of
those 131 cells.
• CED-1 mutants worm (process of phagocytosis is defective) were
mutagenized.
Mutants defective in cell death were obtained (1090 cells in the adult) and
the gene corresponding was called CED-3 (caspase).
Other mutants in which cell death was prevented CED-4.
Worm
EGL-1
Mammals BH3-only
CED-9
CED-4
CED-3
BCL-2-like
Apaf-1-like
Caspase
Cell
death
Apoptosis vs Necrosis
Active mechanism (energy-requiring)
•
Early features (within minutes)
1.
2.
3.
4.
5.
•
mitochondria, lysosomes and cellular
membranes remain intact
Chromatin condensation
DNA fragmentation (multiple of 180
bp) -pyknosisCell shrinkage
Dilatation of ER
•
•
•
•
Affects groups or whole tissue after
damage induced by external stimuli
Energy-independent mechanism
12-24 h
Loss of mitochondria and ER (no
more energy)
Cellular and nuclear swelling and rupture
of cell membrane
Later features (within hours)
Budding of cell membrane
! Release of lysosomial enzymes
Then triggering inflammatory response
Non-specific DNA degradation
apoptotic bodies formation
! No inflammatory response!
APOPTOSIS
Provoking stimuli
Morphological changes
Affected cells
Cell volume
Chromatin
Lysosomes
Mitochondria
Inflammatory response
Cell fate
Molecular changes
Gene activity
Chromosomal DNA
Ca2+ intracellular
Ion pumps
From The biology of cancer, Weinberg
NECROSIS
•Programmed tissues
remodeling
•Genomic damage
•Hypoxia
•....
•Metabolic stresses
•Changes in pH, temperature
•Hypoxia, anoxia
•Injuries
Individual cells
Decreased
Condensed
Unaffected
Normal initially
None
Apoptotic bodies are
phagocytated
Group of cells
Increased
Fragmented
Abnormal
Aberrant morphologically
Marked
Lysis
Required
Cleaved at specific sites
Increased
Functioning
Not needed
Random cleavage
Unaffected
Lost
Apoptosis: Intrinsic and extrinsic pathways
INTRINSIC
(Mitochondrial pathway)
EXTRINSIC
(Death receptor pathway)
Involves release of cytochrome c
(and other proteins) from
mitochondria
Activated by the engagement of
death receptors on cell surface
Activation of caspases
Fesik Nat. Rev. Cancer (2005)
Extrinsic pathway
• Initiated by extracellular signal molecules belonging to the TNF (Tumor Necrosis
Factor) family (TNFα, FAS/CD95 ligand, APO ligand/TRAIL)
• These agonists recognize and activate their corresponding receptor
receptor family such as TNFR1, FAS/CD95, APO2)
(TNF/NGF
• When FASL binds to its receptor FAS, clustering of FAS is triggered and adaptor
proteins are recruited: FADD (FAS-associated death domain) to form a complex called
DISC (death-inducing signaling complex).
DISC recruits and promotes the activation of the initiator procaspase-8.
These activated caspases trigger a caspase cascade activating the executioner
caspases such as caspase-3 and caspase-7 that mediated cell killing.
Alternative name of receptors
Alternative name of ligands
Fas/APO-1/CD95
FasL/CD95L
TNFR1
TNF-α
DR3/APO-3/SWL-1/TRAMP
APO3L
DR4/TRAIL-R1
APO2L/TRAIL
DR5/TRAIL-R2/KILLER
APO2L/TRAIL
From The biology of cancer, Weinberg
MacFarlane et al. EMBO reports (2004)
Intrinsic pathway
Key protagonists:
• Cytochrome C
• Bcl-2 family members
• APAF-1 (apoptotic protease-activating factor 1)
Cytochrome is released from the mitochondrion
mediated by MOMP (mitochondrial outer
membrane permeabilization) and associates with
APAF-1 constituting the apoptosome (the “wheel
of death”). This structure binds to procaspase-9
promoting its activation.
MOMP release also:
• SMAC/DIABLO (IAPs inhibitor)
• HTRA2/OMI (IAPs inhibitor)
• AIF (chromatin condensation)
• Endo G (DNA fragmentation)
Riedl et al. Nat Rev Mol Cell Biol. (2004)
Bcl-2 family proteins
• Bcl-2 family members directly regulate the release of cytochrome c.
• This family contains both pro- and anti-apoptotic proteins.
ANTI-APOPTOTIC Bcl-2; Bcl-XL; Bcl-W; A1; Mcl-1
PRO-APOPTOTIC Bax family (Bax; Bak; Bok)
BH3-only family (Bid; Bim, Bik, Bad, Bmf, Hrk, Noxa; Puma)
The level between pro- and anti- apoptotic proteins determines if cytochrome c is
released from the mitochondrion (BALANCE IS VERY IMPORTANT).
Bcl-2 family proteins
ANTI-APOPTOTIC
Bcl-2 family
BCL-2
BCl-XL
BCL-w
A1
MCL1
PRO-APOPTOTIC
Bax family
BAX
BAK
BOK
BID
BH3-only family
BH= Bcl-2 Homology
TM= transmembrane
BIM
BIK
BAD
BMF
NOXA
PUMA
Apoptosome: “the wheel of death”
Given by the association of APAF-1 and cytochrome c
CARD= Caspase recruitment domain
NBD= Nucleotide binding domain
Adams and Cory Curr Opinion in Cell Biol (2002)
Caspases
Cysteine proteases that cleave after an Asp residue in their substrate.
2 functional categories:
•
•
Initiator caspases (trigger onset of apoptosis by activating the caspase
cascade)
Executioner caspases (undertake the actual work of destroying critical
components of the cell)
– Cleavage of structural components of the cytoskeleton and the nuclear
membrane (actin, cytokeratins and lamins)
– Cause phosphatidylserine to be exposed on the outside of the cellular
membrane (promotion of phagocytosis)
– Inhibit genes that regulate DNA repair during cell cycle (MDM2 and RB)
– Inactivate enzymes responsible for stability, integrity and repair of DNA
(PARP)
– Cleave ICAD (inhibitor of the caspase-activated DNase)
HUMAN CASPASES
CARD= caspase-recruitment domain
DED= death effector domain
L= large catalytic subunit
S= small catalytic subunit
Fuentes-Prior and. Salvesen. Biochem J (2004)
Procaspase-3
Procaspase-8
Procaspase-9
Hengartner Nature (2000)
IAPs (Inhibitors of Apoptosis)
These proteins act inhibiting caspase activity in 2 different ways:
• Direct binding inhibiting the proteolytic activity of caspases
• Marking caspases for ubiquitination and so degradation
Inhibited by SMAC/DIABLO.
Salvesen and Duckett Nat rev mol cell biol (2002)
Deregulation of apoptosis
• Insufficient apoptosis found in cancer or autoimmunity
• Accelerated cell death is found in degenerative diseases and immunodeficiency
Apoptosis and cancer
Tumour cells can acquire resistance to apoptosis by the expression of anti-apoptotic
proteins or by the down-regulation or mutation of pro-apoptotic proteins. Resistance of
tumour cells to apoptosis is an essential feature of cancer development. In fact, this
assumption is confirmed by the finding that deregulated proliferation alone is not
sufficient for tumour formation, but leads to cell death: over-expression of growthpromoting oncogenes, such as c-MYC, sensitizes cells to apoptosis.
Resistance mechanisms:
• Expression of anti-apoptotic proteins (Bcl-2 over-expression in follicular B-cell
lymphoma; over-expression of IAPs in different types of cancers including
neuroblastoma)
• Inactivation of pro-apoptotic genes (BAX mutation; APAF-1 in melanomas)
• Alteration of p53 pathway (p53 mutation)
• Altered survival signalling (alteration of PI3K/Akt pathway- for example PTEN deletion)
Alteration
Mechanism of antiapoptotic action
CASP3 repression
Inactivation of executioner
caspase
Breast carcinomas
p53 mutation
Loss of ability to induce proapoptotic genes
Many types
NF-kB constitutive
activation
Induction of anti-apoptotic
genes
Many types
Mdm2 over-expression
Suppression of p53 levels
Sarcomas
APAF-1 methylation
Loss of proacaspase-9
activation by Cytochrome c
Melanomas
BAX mutation
Loss of pro-apoptotic protein
Colon carcinomas
Bcl-2 over-expression
Closes mitochondrial channels
~ 50% of human tumours
Akt/PKB activation
Phosphorylation and
inactivation of pro-apoptotic
Bcl2-like proteins
Many types
Adapted from The biology of cancer, Weinberg
Types of tumors
p53 and apoptosis
BCL-2
BCL-xL
p53transcriptional
targets
BAX
Direct binding
of p53
NOXA
p53
PUMA
Transcriptional
repression
APAF-1
IAP
Survivin
From The molecular Biology of Cancer Pelengaris and Khan
BCL-2
Apoptosis in the treatment of cancer
An important goal of cancer
drug development should be
to facilitate apoptosis in
neoplastic cells. Drugs that
restore
apoptosis
might
selectively kill cancer cells
that have triggered a death
signal and have become
dependent
on
the
deregulation of apoptosis
pathways.
Strategies already used:
• Administration of death
ligand
• Bcl-2 family inhibitors
• XIAP inhibitors
Fesik Nat Rev Cancer (2005)
„Don't think of death as an ending. Think of it as a really effective
way of cutting down your expenses.”
Woody Allen