Imad Fadl-Elmula

1. APOPTOSIS PROGRAMMED CELL DEATH Imad Fadl-Elmula

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

3. Importance Essential for physiological and developmental process • Embryonic development. • Endocrine-dependent tissue atrophy, • Normal tissue turnover, • Sexual differentiation and clonal selection of immune system.

4. Cells death types There are two distinct modes of cell death: 1. Necrosis Results from toxins and ischemia injury by agents Affects cells in groups rather than singly Evokes exudative inflammation when it develops. 2. Apoptosis The cells undergo cytoplasmic shrinkage, nuclear condensation and/or fragmentation. membrane blebbing. Moreover, inflammation does not occur while it is evident in necrosis. the second p is pronounced as in sym-ptom.

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

6. What we will gain? The understanding of discrete apoptotic pathways will obviously contribute not only to the explanations of the development of cancer, but also the development of new strategies toward the prevention and therapy of the disease.

7. Genetic Alterations

14. Keeping apoptosis under control. Activation mechanism Negative regulator autoactivated protease cascade ced-9 and bcl-2 family proteins

15. Cell Death Apoptosis switch

16. How Apoptosis occurs Cell Death Apoptosis switch

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

22. 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

24. Lonising radiation effect DNA damage and induces P53 expression I-V Evolutionary conserved regions transactivation Oligomerisation Binding sites on transcribed protein of gene Mutation hotspot zones 5-terminus 3-terminus Gene X P53 Gene 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 phase to effect DNA repair mechanisms Cell cycles normally if repear of gene X is successful Apoptosis occurs if repair of gene X is unsuccessful prevent continuation of genetic defect and possible development of tumour cell population

25. Death Receptor-ligand interactions Apaf-1 Cytochrome C Regulated by cytokines and hormones Mitochondria Cell Injury Initiator Caspases Execution Caspases DNA Fragmentation Nucleus

27. 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.

28. 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

29. APOPTOSIS: Overall scheme • General 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

30. APOPTOSIS: Disease related proteins • Autoimmune lymphoproliferative syndrome • Tumor necrosis factor ligandsuperfamily, 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

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

32. There are two ways in which cells die: • they are killed by injurious agents • they are induced to commit suicide

33. 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

34. 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.)

35. 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

36. 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

38. 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).

39. The modes of cell death Necrosis A pathological response to cellular injury. Chromatin clumps Mitochondria swell and rupture Plasma membrane lyses Cell contents spill out General inflammatory response is triggered. Apoptosis Apoptosis: a normal physiological response to specific suicide signals, or lack of survival signals.

40. 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.