ApoptosisApoptosis

Book reading club: 9th February 2007

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 process in:

• Embryonic development

• Normal cellular homeostasis

Apoptosis: the functions

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

Mammals

EGL-1

BH3-only

CED-9

BCL-2-like

CED-4

Apaf-1-like

CED-3

Caspase

CellCell deathdeath

Apoptosis vs Necrosis

• Early features (within minutes)1. mitochondria, lysosomes and cellular

membranes remain intact2. Chromatin condensation 3. DNA fragmentation (multiple of 180 bp) -

pyknosis-4. Cell shrinkage 5. Dilatation of ER

• Later features (within hours)Budding of cell membrane

apoptotic bodies formation

! No inflammatory response!

• 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

! Release of lysosomial enzymes Then triggering inflammatory responseNon-specific DNA degradation

Active mechanism (energy-requiring)

APOPTOSISAPOPTOSIS NECROSISNECROSIS

Provoking stimuli •Programmed tissues remodeling•Genomic damage•Hypoxia•....

•Metabolic stresses•Changes in pH, temperature•Hypoxia, anoxia•Injuries

Morphological changes

Affected cells

Cell volume

Chromatin

Lysosomes

Mitochondria

Inflammatory response

Cell fate

Individual cells

Decreased

Condensed

Unaffected

Normal initially

None

Apoptotic bodies are phagocytated

Group of cells

Increased

Fragmented

Abnormal

Aberrant morphologically

Marked

Lysis

Molecular changes

Gene activity

Chromosomal DNA

Ca2+ intracellular

Ion pumps

Required

Cleaved at specific sites

Increased

Functioning

Not needed

Random cleavage

Unaffected

Lost

From The biology of cancer, Weinberg

Apoptosis: Intrinsic and extrinsic pathways

EXTRINSIC

(Death receptor pathway)

Activated by the engagement of death receptors on cell surface

INTRINSIC

(Mitochondrial pathway)

Involves release of cytochrome c (and other proteins) from

mitochondria

Activation of caspasesActivation 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 (TNF/NGF receptor family such as TNFR1, FAS/CD95, APO2)

• 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 receptorsAlternative name of receptors Alternative name of ligandsAlternative 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).

ANTI-APOPTOTIC

Bcl-2 family

Bax family

BH3-only family

PRO-APOPTOTIC

BCL-2BCl-XL

BCL-wA1MCL1

BAXBAKBOK

BID

BIMBIKBADBMFNOXAPUMA

Bcl-2 family proteins

BH= Bcl-2 HomologyTM= transmembrane

Apoptosome: “the wheel of death”

Adams and Cory Curr Opinion in Cell Biol (2002)

CARD= Caspase recruitment domainNBD= Nucleotide binding domain

Given by the association of APAF-1 and cytochrome c

Caspases

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)

Cysteine proteases that cleave after an Asp residue in their substrate.

Fuentes-Prior and. Salvesen. Biochem J (2004)

HUMAN CASPASES

CARD= caspase-recruitment domainDED= death effector domainL= large catalytic subunitS= small catalytic subunit

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)

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 growth-promoting 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)

Deregulation of apoptosis• Insufficient apoptosis found in cancer or autoimmunity• Accelerated cell death is found in degenerative diseases and immunodeficiency

AlterationAlteration Mechanism of anti-Mechanism of anti-apoptotic actionapoptotic action Types of tumorsTypes of tumors

CASP3 repressionInactivation of executioner caspase

Breast carcinomas

p53 mutationLoss of ability to induce pro-apoptotic 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 activationPhosphorylation and inactivation of pro-apoptotic Bcl2-like proteins

Many types

Adapted from The biology of cancer, Weinberg

p53 and apoptosis

p53

BAX

NOXA

PUMA

APAF-1

BCL-2

BCL-2

BCL-xL

IAP

Survivin

p53-transcriptional

targets

Direct binding of p53

Transcriptional repression

From The molecular Biology of Cancer Pelengaris and Khan

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