History History Described first by Kerr et al., (1972).Described first by Kerr et al., (1972).Apoptosis derived from Greek Apoptosis derived from Greek "falling off""falling off" (as (asfor autumn leaves).for autumn leaves).

ImportanceImportance

Essential for physiological and developmental processEssential for physiological and developmental process Embryonic development.Embryonic development. Endocrine-dependent tissue atrophy,Endocrine-dependent tissue atrophy, Normal tissue turnover, Normal tissue turnover, Sexual differentiation and clonalSexual differentiation and clonal

selection of immune system.selection of immune system.

Cells death typesCells death types

There are two distinct modes of cell death:There are two distinct modes of cell death:1. Necrosis1. Necrosis

Results from toxins and ischemia injury by agentsResults from toxins and ischemia injury by agentsAffects cells in groups rather than singlyAffects cells in groups rather than singlyEvokes exudative inflammation when it develops. Evokes exudative inflammation when it develops.

2. Apoptosis2. ApoptosisThe cells undergo cytoplasmic shrinkage, The cells undergo cytoplasmic shrinkage, nuclear condensation and/or fragmentation. nuclear condensation and/or fragmentation. membrane blebbing. Moreover, membrane blebbing. Moreover,

inflammation does not occur while it is evident in necrosis. inflammation does not occur while it is evident in necrosis. the second the second pp is pronounced as in sym- is pronounced as in sym-ptptom. om.

Apoptosis and CancerApoptosis and Cancer

Apoptosis of DNA-damaged cells may Apoptosis of DNA-damaged cells may constitute a physiological antineoplastic constitute a physiological antineoplastic mechanism to protect the organism from mechanism to protect the organism from cancer development by eliminating cells cancer development by eliminating cells that might otherwise replicate the that might otherwise replicate the damaged DNA and lead to mutations and damaged DNA and lead to mutations and to cancer to cancer

What we will gainWhat we will gain??

The understanding of discrete apoptoticThe understanding of discrete apoptotic

pathways will obviously contribute not onlypathways will obviously contribute not only

to the explanations of the development ofto the explanations of the development of

cancer, but also the development of newcancer, but also the development of new

strategies toward the prevention and therapystrategies toward the prevention and therapy

of the disease.of the disease.

Genetic Alterations

Keeping apoptosis under control.

ced-9 and bcl-2 family proteins

Negative regulatorActivation mechanism

autoactivated protease cascade

Apoptosis switch Cell Death

Apoptosis switchApoptosis switch Cell Cell DeathDeath

the cell swells components of the cell (e.g. the DNA in the nucleus) stay pretty much the same the basic ithe cell disintergrates, leaving a mess behind nternaltends to result from some some external force (e.g. an injury or infection

the cell swells the basic internal components of the cell (e.g. the DNA in the nucleus)

stay pretty much the same the cell disintergrates, leaving a mess behind

tends to result from some some external force (e.g. an injury or infection

Cancer cells do not arrest in G1 or G2 phase and replication of damaged DNA results in an accumulation of mutations

Normal cells arrest in G1 or G2 phaseto effect DNA repair mechanisms

Cell cycles normallyif repear of gene X is successful

Apoptosis occurs if repair of gene X is unsuccessfulprevent continuation of genetic defect and possible development of tumour cell population

P53 GeneGene X

Lonising radiation effect DNAdamage and inducesP53 expression

I-V Evolutionary conserved regionstransactivation

Oligomerisation

Binding sites on transcribed protein of gene

Mutation hotspot zones

3-terminus5-terminus

Death Receptor-ligand interactionsDeath Receptor-ligand interactionsApaf-1Apaf-1Cytochrome CCytochrome C

Regulated byRegulated bycytokines andcytokines and

hormoneshormones

MitochondriaMitochondria

Cell InjuryCell Injury

NucleusNucleus

DNADNAFragmentationFragmentation

Initiator CaspasesInitiator Caspases

Execution CaspasesExecution Caspases

• Cell death programmes, far from being sinister, are essential to proper growth and development. In order to make the spaces between your fingers, for example, certain cells had to die and be removed during your development.

• Apoptosis is particularly interesting to humans because problems with cell death not going on as planned are implicated in diseases like Alzheimer's, and because the ability to target and kill cells selectively would be of great benefit in the treatement of diseases like cancers.

• But before we get too excited about this, does apoptosis occur in single celled organisms?

• it has been documented in bacteria (Yarmolinsky 1995) • it has been documented in protozoans (Welburn et al. 1997) • I have published some evidence for it in phytoplankton (Berges and

Falkowski 1998) • But why would a unicellular organism kill itself?• in a time of starvation, it would liberate nutrients for neighbouring

(genetically identical) cells (altruism; Yarmolinsky 1995) • it is a response to disease, i.e. a way to prevent a viral infection from

spreading to other cells (sacrifice in the face of foes; Mittler and Lam 1996) • it reflect a degree of 'social organisation' among unicellular populations (Raff

1992) • there is no reason: it is just an obscure pathway that developed in

unicellular organisms and simply persisted until multicellular organisms arose, when it became useful

APOPTOSIS: Overall schemeGeneral Causes External Exogenous oxidative stress: Ischemia (Mild) Excitotoxins: Glutamate; Ion fluxes Trophic factors: Reduced receptor occupancy Stimuli: TNF-a; Corticosteroids (Lymphoid system) Toxins: b-Amyloid Internal Toxic substances: Mutated SOD1 Byproducts of cell metabolism Cell signals Cell stress Mitochondrial permeability increased Release of cytochrome c Extracellular signal: Ligand-Receptor

Caspase activation Expression of "Death" genes: Reversible Terminal apoptotic changes: Irreversible Cell Death

External link :R&D

• APOPTOSIS: Disease related proteins• Autoimmune lymphoproliferative syndrome

– Tumor necrosis factor ligand superfamily, Member 6 (TNFSF6; CD95L; CD178) : Also Systemic lupus erythematosus, susceptibility

– Tumor necrosis factor receptor superfamily, Member 6 (TNFRSF6; FAS antigen; CD95) – Caspase 8

• Lymphadenopathy, splenomegaly & defective CD95-induced apoptosis of peripheral blood lymphocytes • Recurrent sinopulmonary & herpes simplex virus infections • Poor responses to immunization • Defects in activation of T lymphocytes, B lymphocytes, & natural killer cells leading to immunodeficiency

– Caspase 10 (Apoptosis-related cysteine protease) • Neoplasms

– Tumor protein p53 • Li-Fraumeni syndrome • Other neoplasms: Pancreatic; Hepatocellular; Histiocytoma; Osteosarcoma; Breast; Nasopharyngeal

– Tumor necrosis factor receptor superfamily, Member 6 (TNFRSF6; FAS antigen; CD95) : Squamous cell – Tumor necrosis factor receptor superfamily, Member 10B (TNFRSF10B) : Squamous cell – BCL2-associated X protein (BAX) : T-Cell acute lymphoblastic leukemia; Colorectal

• Mouse disorders – Caspase 9 : Knockout

• Enlarged & malformed cerebrum: Caused by reduced apoptosis during brain development

For every cell, there is a time to live and a time to die .

• There are two ways in which cells die:

• they are killed by injurious agents

• they are induced to commit suicide

• Death by injury• Cells that are damaged by injury, such as by • mechanical damage • exposure to toxic chemicals• undergo a characteristic series of changes: • they (and their organelles like mitochondria)

swell (because the ability of the plasma membrane to control the passage of ions and water is disrupted)

• the cell contents leak out, leading to • inflammation of surrounding tissues

• Death by suicide• Cells that are induced to commit suicide: • shrink • have their mitochondria break down with the release of cytochrome c • develop bubble-like blebs on their surface • have the chromatin (DNA and protein) in their nucleus degraded • break into small, membrane-wrapped, fragments • The phospholipid phosphatidylserine, which is normally hidden within the plasma

membrane is exposed on the surface. • This is bound by receptors on phagocytic cells like macrophages and dendritic cells

which then engulf the cell fragments. • The phagocytic cells secrete cytokines that inhibit inflammation.• The pattern of events in death by suicide is so orderly that the process is often called

programmed cell death or PCD. The cellular machinery of programmed cell death turns out to be as intrinsic to the cell as, say, mitosis. Programmed cell death is also called apoptosis. (There is no consensus yet on how to pronounce it; some say  APE oh TOE sis; some say  uh POP tuh sis.)

• Why should a cell commit suicide?• There are two different reasons. 1. Programmed cell death is as needed for proper development as mitosis

is. • Examples: • The resorption of the tadpole tail at the time of its metamorphosis into a frog occurs by apoptosis. • The formation of the fingers and toes of the fetus requires the removal, by apoptosis, of the tissue between them. • The sloughing off of the inner lining of the uterus (the endometrium) at the start of menstruation occurs by

apoptosis. • The formation of the proper connections (synapses) between neurons in the brain requires that surplus cells be

eliminated by apoptosis• 2. Programmed cell death is needed to destroy cells that represent a threat to the integrity of the

organism.• Examples: • Cells infected with viruses

– One of the methods by which cytotoxic T lymphocytes (CTLs) kill virus-infected cells is by inducing apoptosis [diagram of the mechanism]. (And some viruses mount countermeasures to thwart it.)

• Cells of the immune system – As cell-mediated immune responses wane, the effector cells must be removed to prevent them from attacking body constituents.

CTLs induce apoptosis in each other and even in themselves. Defects in the apoptotic machinery is associated with autoimmune diseases such as lupus erythematosus and rheumatoid arthritis.

• Cells with DNA damage – Damage to its genome can cause a cell

– to disrupt proper embryonic development leading to birth defects – to become cancerous.

– Cells respond to DNA damage by increasing their production of p53. p53 is a potent inducer of apoptosis. Is it any wonder that mutations in the p53 gene, producing a defective protein, are so often found in cancer cells (that represent a lethal threat to the organism if permitted to live)?

• Cancer cells – Radiation and chemicals used in cancer therapy induce apoptosis in some types of cancer cells

• The Mechanisms of Apoptosis• There are 3 different mechanisms by which a cell commits suicide by apoptosis. • one generated by signals arising within the cell • another triggered by death activators binding to receptors at the cell surface.

– TNF-a – Lymphotoxin – Fas ligand (FasL)

• a third that may be triggered by dangerous reactive oxygen species. • 1. Apoptosis triggered by internal signals: the intrinsic or mitochondrial pathway• • In a healthy cell, the outer membranes of its mitochondria express the protein Bcl-2 on their surface. • Bcl-2 is bound to a molecule of the protein Apaf-1. • Internal damage to the cell (e.g., from reactive oxygen species) causes

– Bcl-2 to release Apaf-1; – a related protein, Bax, to penetrate mitochondrial membranes, causing cytochrome c to leak out.

• The released cytochrome c and Apaf-1 bind to molecules of caspase 9. • The resulting complex of

– cytochrome c – Apaf-1 – caspase 9 – (and ATP)

• is called the apoptosome. • These aggregate in the cytosol. • Caspase 9 is one of a family of over a dozen caspases. They are all proteases. They get their name because

they cleave proteins - mostly each other - at aspartic acid (Asp) residues). • Caspase 9 cleaves and, in so doing, activates other caspases. • The sequential activation of one caspase by another creates an expanding cascade of proteolytic activity (rather

like that in blood clotting and complement activation) which leads to – digestion of structural proteins in the cytoplasm – degradation of chromosomal DNA and

• phagocytosis of the cell

• Apoptosis and Cancer• Some cancer-causing viruses use tricks to prevent apoptosis of the cells they have transformed. • Several human papilloma viruses (HPV) have been implicated in causing cervical cancer. One of them

produces a protein (E6) that binds and inactivates the apoptosis promoter p53. • Epstein-Barr Virus (EBV), the cause of mononucleosis and a cause of Burkitt's lymphoma

– produces a protein similar to Bcl-2 – produces another protein that causes the cell to increase its own production of Bcl-2. Both these

actions make the cell more resistant to apoptosis (thus enabling the cancer cell to continue to proliferate).

• Even cancer cells produced without the participation of viruses may have tricks to avoid apoptosis. • Some B-cell leukemias and lymphomas express high levels of Bcl-2, thus blocking apoptotic signals they

may receive. The high levels result from a translocation of the BCL-2 gene into an enhancer region for antibody production. [Discussion].

• Melanoma (the most dangerous type of skin cancer) cells avoid apoptosis by inhibiting the expression of the gene encoding Apaf-1.

• Some cancer cells, especially lung and colon cancer cells, secrete elevated levels of a soluble "decoy" molecule that binds to FasL, plugging it up so it cannot bind Fas. Thus, cytotoxic T cells (CTL) cannot kill the cancer cells by the mechanism shown above.

• Other cancer cells express high levels of FasL, and can kill any cytotoxic T cells (CTL) that try to kill them because CTL also express Fas (but are protected from their own FasL).

The modes of cell deathThe modes of cell deathNecrosisNecrosisA pathological response to cellular injury.

Chromatin clumps

Mitochondria swell and rupture

Plasma membrane lyses

Cell contents spill out

General inflammatory response is triggered.

ApoptosisApoptosis: a normal physiological response to specific suicide

signals, or lack of survival signals.

When?In embryonic and fetal development: • Tissue developmental programs which control sculpting of embryonic

form. • Developmental organization of the nervous system; • Elimination of self-reactive components of the immune system.

In the adult: • On stimulation by T-lymphocytes. • In response to DNA damage or abnormality, e.g. by radiation or viral

infection or transformation. • In certain organs and tissues, on withdrawal of supporting hormones In addition, there are often apoptotic centres in tumours, accounting for

the paradox of slow gross enlargement in the face of rapid cell proliferation, and the rare spontaneous remission.