2 3. Morphological features of apoptosis Content1. The development of the term apoptosis2. The significance of apoptosis3. Morphological features of apoptosis4. Molecular mechanisms of apoptosis signalling pathways5. Disease as a consequence of dysregulated apoptosis
4 That cell death is a completely normal process in living organisms was already discovered by scientists more than 100 years ago. The German scientist Carl Vogt was first to describe the principle of apoptosis in 1842.since the mid-nineteenth century, many observations have indicated that cell death plays a considerable role during physiological processes of multicellular organisms, particularly during embryogenesis and metamorphosis [Gluecksmann, 1951; Lockshin, 2001].The term programmed cell death was introduced in 1964, proposing that cell death during development is not of accidential nature but follows a sequence of controlled steps leading to locally and temporally defined self-destruction [Lockshin, 1964].
5 the term apoptosis had been coined in order to describe the morphological processes leading to controlled cellular self-destruction and was first introduced in a publication by Kerr, Wyllie and Currie [Kerr, 1972].Apoptosis is of greek origin, having the meaning "falling off or dropping off", in analogy to leaves falling off trees or petals dropping off flowers[Leist, 2001a].
7 Physiological roles of apoptosis Development:Figure 1. Proper development of multicellular organisms depends on the elimination of selected cells through apoptosis. As a tadpole becomes a frog it deletes its tail cells. Human embryos are also thought to use apoptosis to remove webbing between digits [Jared Schneidman].
8 Figure 2. The effect of lack of programmed cell death (Specifically apoptosis) on the toes of a human. A mutation caused the middle two toes to remain connected.
9 Homeostasis:Also cells of an adult organism constantly undergo physiological cell death which must be balanced with proliferation in order to maintain homeostasis in terms of constant cell numbers[Rathmell, 2002].
10 Skin: Skin cells begin life in the deepest layers and then migrate to the surface in layers, undergoing apoptosis along the way. The resulting dead cell layer forms the protective outer skin layer, called the epidermis.Uterus: The cells of the uterine wall die and are sloughed off during menstruation. This action is accomplished by apoptosis.Thymus: T Lymphocytes (or T-cells) are white blood cells that are critical components of the immune system; these immune cells mature in the thymus gland (located in the upper chest area just below the neck). T-cells that would be ineffective or that would attack the body's own tissues commit suicide before they have the chance to enter the blood stream.Figure 2 . Apoptotic Cells in the adult - Virtually all tissues harbor apoptotic cells at one time or another. The cells usually commit suicide for the greater good of the body.
11 Cell termination:Apoptosis can occur when a cell is damaged beyond repair, infected with a virus, or undergoing stress conditions such as starvation. DNA damage from ionizing radiation or toxic chemicals can also induce apoptosis via the actions of the tumour-suppressing gene p53. The "decision" for apoptosis can come from the cell itself, from the surrounding tissue, or from a cell that is part of the immune system. In these cases apoptosis functions to remove the damaged cell, preventing it from sapping further nutrients from the organism, or to prevent the spread of viral infection.
12 Apoptosis also plays a role in preventing cancer; if a cell is unable to undergo apoptosis, due to mutation or biochemical inhibition, it can continue dividing and develop into a tumour. For example, infection by papillomaviruses causes a viral gene to interfere with the cell's p53 protein, an important member of the apoptotic pathway. This interference in the apoptotic capability of the cell plays a critical role in the development of cervical cancer.
13 When normal cells are damaged beyond repair, they are eliminated by apoptosis (A). Cancer cells avoid apoptosis and continue to multiply in an unregulated manner (B).
15 Apoptotic versus necrotic morphology [Saraste, 2000].
16 4. Molecular mechanisms of apoptosis signalling pathways 4.1 Various death signals activate common signalling pathways4.2 Caspases are central initiators and executioners of apoptosis4.3 Extrinsic apoptosis pathways of type I and type II4.4 Mitochondria as central regulators of intrinsic apoptosis pathways
17 4.1 Various death signals activate common signalling pathways Apoptosis is a tightly regulated and at the same time highly efficient cell death program which requires the interplay of a multitude of factors. The components of the apoptotic signalling network are genetically encoded and are considered to be usually in place in a nucleated cell ready to be activated by a death-inducing stimulus [Ishizaki, 1995; Weil, 1996].
18 developmental death signals DNA (normal) DNA (damaged) cytotoxicDNA repairactivate a common cell death machineryapoptotic cell death
19 4.2 Caspases are central initiators and executioners of apoptosis The term caspases is derived from cysteine-dependent aspartate-specific proteases.So far, 7 different caspases have been identified in Drosophila, and 14 different members of the caspase-family have been described in mammals, with caspase-11 and caspase–12 only identified in the mouse [Denault, 2002; Richardson, 2002].They are : 1,2,3,4,5,6,7,8,9,10,11,12,pro-1L-1B, pro-1L-18
20 The proapoptotic caspases can be divided: into the group of initiator caspases including procaspases-2, -8, -9 and –10, andinto the group of executioner caspases including procaspases-3, -6, and –7.
21 the initiator caspases are recruited to and activated at death inducing signalling complexes either in response to the ligation of cell surface death receptors (extrinsic apoptosis pathways) or in response to signals originating from inside the cell (intrinsic apoptosis pathways).
22 extrinsic apoptosis pathways (DISC): when the procapase-8 is mediated to the receptor complex which called the death-inducing signaling complex(DISC).EX: Receptor-mediated caspase activation at the DISC[Earnshaw, 1999].
23 Intrinsic apoptosis pathways EX: Caspase activation at the apoptosome
24 4.3 Extrinsic apoptosis pathways of type I and type II Cells harboring the capacity to induce such direct and mainly caspase-dependent apoptosis pathways were classified to belong to the so called type I cells [Scaffidi, 1998].In type II cells, the signal coming from the activated receptor does not generate a caspase signalling cascade strong enough for execution of cell death on its own.
26 4.4 Mitochondria as central regulators of intrinsic apoptosis pathways DNA damage, oxidative stressmitochondrial inner transmembrane potential (Dy)permeability transition (PT)of the inner mitochondrial membrane permeability to solutes(swelling)the outer mitochondrial membrane ruptureReleased proteins proapoptotic include cytochrome c
27 Regulation of apoptosis by the Bcl-2 family BH3: required for the activation proapoptotic Bax/Bak function and by the antiapoptitic Bcl-2/BclXL they strongly bind to it so they stay under control.
28 The link between the caspase signalling cascade and the mitochondria is provided by the Bcl-2 family member Bid. Bid is cleaved by caspase-8 and in its truncated form (tBID) translocates to the mitochondria where it acts in concert with the proapoptotic Bcl-2 family members Bax and Bak to induce the release of cytochrome c and other mitochondrial proapoptotic factors into the cytosol [Luo, 1998]).
29 The bisphosphonate zoledronic acid inhibits the growth of HCT-116 colon carcinoma cells and induces tumor cell apoptosis.Apoptosis Apr 25;Authors: Sewing L, Steinberg F, Schmidt H, Göke RBesides its preventive action on bone resorption the third generation bisphosphonate zoledronic acid (ZOL) has been shown to display potent inhibitory action on the formation of bone metastases of various human cancers. Recent research also indicates an antitumoral effect on primary tumors and visceral metastases. Here we investigate for the first time the effect of ZOL on the human colon carcinoma cell line HCT-116. ZOL strongly inhibited the proliferation and soft agar colony formation of HCT-116 cells and caused a G1 cell cycle arrest in a population of ZOL treated cells
30 . This cell cycle arrest was accompanied by an induction of apoptosis via a caspase dependent mechanism. Activation of Caspases 3, 7, 8 and 9, cleavage of PARP as well as the release of cytochrome C into the cytosol were detected in HCT-116 cells treated with low micromolar concentrations of ZOL. The induction of the mitochondrial pathway of apoptosis was accompanied by a translocation of Bax into the mitochondria, Bid activation and a decrease of overall Bcl-2 expression. We also detected a cytosolic increase of apoptosis inducing factor (AIF), a trigger of caspase-independent apoptosis. Taken together, our data indicate a potent antitumoral and apoptosis inducing effect of ZOL on HCT-116 colon carcinoma cells.PMID: [PubMed - as supplied by publisher]
32 BAX and BAK Regulation of Endoplasmic Reticulum Ca2+: A Control Point for Apoptosis BAX and BAK are "multidomain" proapoptotic proteins that initiate mitochondrial dysfunction but also localize to the endoplasmic reticulum (ER). Mouse embryonic fibroblasts deficient for BAX and BAK (DKO cells) were found to have a reduced resting concentration of calcium in the ER ([Ca2+]er) that results in decreased uptake of Ca2+ by mitochondria after Ca2+ release from the ER.
33 Expression of SERCA (sarcoplasmic-endoplasmic reticulum Ca2+ adenosine triphosphatase) corrected [Ca2+]er and mitochondrial Ca2+ uptake in DKO cells, restoring apoptotic death in response to agents that release Ca2+ from intracellular stores (such as arachidonic acid, C2-ceramide, and oxidative stress).In contrast, targeting of BAX to mitochondria selectively restored apoptosis to "BH3-only" signals. A third set of stimuli, including many intrinsic signals, required both ER-released Ca2+ and the presence of mitochondrial BAX or BAK to fully restore apoptosis. Thus, BAX and BAK operate in both the ER and mitochondria as an essential gateway for selected apoptotic signal
34 5. Disease as a consequence of dysregulated apoptosis
35 In the adult human body several hundred thousand cells are produced every second by mitosis, and a similar number die by apoptosis for the maintenance of homeostasis and for specific tasks such as the regulation of immune cell selection and activity [Fadeel, 1999b].
36 Dysregulation of apoptotic signalling can play a primary or secondary role in various diseases with insufficient apoptosis leading to e.g. cancer, autoimmunity, persistent infections (failure to eradicate infected cells), whereas excessive apoptosis contributes to e.g. neurodegeneration (Alzheimers’ disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis), autoimmunity (uncontrolled apoptosis induction in specific organs), AIDS (depletion of T lymphocytes), and ischaemia (stroke, myocardial infarction) [Reed, 2002].
37 Malfunction of the death machinery results from the mutation of genes that code for factors directly or indirectly involved in the initiation, mediation, or execution of apoptosis, and several mutations in apoptosis genes have been identified as a causing or contributing factor in human diseases [Mullauer, 2001].