1. Anatomy and Human Biology 313-2005 Arun Dharmarajan Professor School of Anatomy & Human Biology The University of Western Australia, Perth, Australia

2. Relevance of Apoptosis in Health and Disease Apoptosis is a systematic and genetically programmedprocess. Apoptosis is involved in tissue homeostasis and cell differentiation. Apoptosis is directly involved in degenerative diseases, autoimmune disorders, viral diseases, cancer, and reproductive disorders.

3. Apoptosis Timeline 1842: Vogt recognised a form of Physiological cell death. 1855: Flemming used the term “chromatolysis” 1951: Gluckmann described the morphological basis of apoptosis. 1960: Lockshin-programmed cell death 1972: Wyllie and Kerr- Apoptosis 1985: Horvitz-apoptosis determined by several genes (The Terminators) The Good (blocks); The Bad (executes); The Ugly (activator of apoptosis) These genes are highly conserved throughtout evolution. In Man, there are over 21 Goods (bcl-2 family), 14 Bads (the caspases), BUT only one Ugly (Apaf-1).

4. 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. “Once we are in the land of the living, we will eventually die” This is true not only for human beings but also for the cells that make up our bodies APOPTOSIS

5. Death by Injury Cells that are damaged by injury, such as by mechanical damage/ exposure to toxic chemicals 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.mitochondriainflammation

6. Apoptosis How to die…. A discrete, active and energy dependent form of cell death. Executed roughly 20 times as rapidly as mitosis. Several characteristic morphological features; Chromatin condensation, Membrane blebbing, DNA fragmentation (180bp multiples), No inflammatory response.

7. APOPTOSIS Death by suicide Cells that are induced to commit suicide: shrink; develop bubble-like blebs on their surface; have the chromatin (DNA and protein) in their nucleus degraded;chromatin have their mitochondria break down with the release of cytochrome c; break into small, membrane-wrapped, fragments. cytochrome c The phospholipid phosphatidylserine, which is normally hidden within the plasma membrane, is exposed on the surface.phospholipid This is bound by receptors on phagocytic cells like macrophages and dendritic cells which then engulf the cell fragments.phagocyticmacrophagesdendritic cells The phagocytic cells secrete cytokines that inhibit inflammation (e.g., IL-10 and TGF-β)cytokinesinflammationIL-10 and TGF-β APOPTOSIS

8. Why should a cell commit suicide? There are two different reasons. 1.Programmed cell death is as needed for proper development as mitosis is. 2.Programmed cell death is needed to destroy cells that represent a threat to the integrity of the organism. e.g Cells infected with viruses Cells of the immune system Cells with DNA damage Cancer cells

9. What makes a cell decide to commit suicide? The balance between: the withdrawal of positive signals; that is, signals needed for continued survival e.g Growth Factors/Hormones the receipt of negative signals e.g. UV/oxidants/ X-Rays/Chemo Death activators: TNF/ Fas/Lymphotoxin APOPTOSIS

10. 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; anotheranother triggered by death activators binding to receptors at the cell surface: TNF-α Lymphotoxin Fas ligand (FasL) A third that may be triggered by dangerous reactive oxygen species.thirdreactive oxygen species APOPTOSIS

11. 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 ("apoptotic protease activating factor-1". Bcl-2 Internal damage to the cell (e.g., from reactive oxygen species) causesreactive oxygen species Bcl-2 to release Apaf-1; a related protein, Bax, to penetrate mitochondrial membranes, causing cytochrome c to leak out.cytochrome c 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)ATP is called the apoptosome. APOPTOSIS

12. 2. Apoptosis triggered by external signals: the extrinsic or death receptor pathway Fas and the TNF receptor are integral membrane proteins with theirintegral membrane proteins receptor domains exposed at the surface of the cell binding of the complementary death activator (FasL and TNF respectively) transmits a signal to the cytoplasm that leads to activation of caspase 8 caspase 8 (like caspase 9) initiates a cascade of caspase activation leading to phagocytosis of the cell. APOPTOSIS

13. Apoptosis-Inducing Factor (AIF) Neurons, and perhaps other cells, have another way to self-destruct that — unlike the two paths described above — does not use caspases. Apoptosis-inducing factor (AIF) is a protein that is normally located in the intermembrane space of mitochondria.intermembrane space of mitochondria When the cell receives a signal telling it that it is time to die, AIF is released from the mitochondria (like the release of cytochrome c in the first pathway); migrates into the nucleus; binds to DNA, which triggers the destruction of the DNA and cell death. first pathway APOPTOSIS

14. Apoptosis and Cancer Some cancer-causing viruses use tricks to prevent apoptosis of the cells they have transformed.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.human papilloma virusesE6p53 Epstein-Barr Virus (EBV), the cause of mononucleosis and a cause of Burkitt's lymphoma produces a protein similar to Bcl-2 Burkitt's lymphoma 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). APOPTOSIS

15. 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. 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.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).

16. 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; anotheranother triggered by death activators binding to receptors at the cell surface: TNF-α Lymphotoxin Fas ligand (FasL) A third that may be triggered by dangerous reactive oxygen species.thirdreactive oxygen species APOPTOSIS

17. TNF Signal Transduction CERAMIDE P38 MAPK CLEAVAGE OF DEATH SUBSTRATES APOPTOTIC PHENOTYPE TNFR2 TNFR1 ERK 1&2 SPHINGOSINE CELL SURVIVAL CASPASES JNK NF-B NITRIC OXIDE

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20. Assays that Measure DNA Fragmentation (HMW DNA, DNA Content) Assays that Examine Chromatin Morphology Assays that Measure DNA Strand Breaks (Nicks) and DNA Fragmentation (Staggered DNA Ends) Assays that Detect Phosphatidylserine on the Surface of Apoptotic Cells Assays that Measure Plasma Membrane Damage/Leakage Comet Assay APOPTOSIS

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31. METHOD Cultured cells or frozen section Embed cells/section in agarose Lyse cell membrane Equilibrate in electrophoresis buffer Electrophoresis Stain and view

32. Comet Assay Visual measure of DNA damage Cells lysed, electrophoresed, stained Image produced resemble comet Advantages 1. Distinguish necrosis from apoptosis 2. Identify cell types undergoing apoptosis