Apoptosis Book reading club: 9 th 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 Celldeath

Apoptosis vs Necrosis Early features (within minutes) 1.mitochondria, lysosomes and cellular membranes remain intact 2.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 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 response Non-specific DNA degradation Active mechanism (energy-requiring)

APOPTOSISNECROSIS Provoking stimuliProgrammed 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 Ca 2+ 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 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 receptors Alternative name of ligands Fas/APO-1/CD95FasL/CD95L TNFR1TNF-α DR3/APO-3/SWL-1/TRAMPAPO3L DR4/TRAIL-R1APO2L/TRAIL DR5/TRAIL-R2/KILLERAPO2L/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-APOPTOTICBcl-2; Bcl-X L ; Bcl-W; A1; Mcl-1 PRO-APOPTOTICBax 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-2 BCl-X L BCL-w A1 MCL1 BAX BAK BOK BID BIM BIK BAD BMF NOXA PUMA Bcl-2 family proteins BH= Bcl-2 Homology TM= transmembrane

Apoptosome: “the wheel of death” Adams and Cory Curr Opinion in Cell Biol (2002) CARD= Caspase recruitment domain NBD= 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 domain DED= death effector domain L= large catalytic subunit S= 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

Alteration Mechanism of anti- apoptotic action Types of tumors CASP3 repression Inactivation of executioner caspase Breast carcinomas p53 mutation Loss of ability to induce pro- apoptotic genes Many types NF-kB constitutive activation Induction of anti-apoptotic genes Many types Mdm2 over-expressionSuppression of p53 levelsSarcomas APAF-1 methylation Loss of proacaspase-9 activation by Cytochrome c Melanomas BAX mutationLoss of pro-apoptotic proteinColon carcinomas Bcl-2 over-expressionCloses 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

p53 and apoptosis p53 BAX NOXA PUMA APAF-1 BCL-2 BCL-x L 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