1. PROGRAMMED CELL DEATH ( APOPTOSIS )
Prof Dr Turgay İSBİR

2. APOPTOSIS
Cell proliferation must be balanced by an appropriate process of cell death in order to maintain tissue homeostasis. Physologic cell death is a genetic program pathway and şs called APOPTOSIS. Apoptosis is derived from GREEK and means dropping of leaves or petals from flowers. This word was coined in 1972 to describe a type of cell death that exhibits very characteristic morphological properties. This cascade of events includes:
Condesantion of chromatin
Breaking up of the nucleus
Bleeding of the plasma membrane
Fragmentation of the cell into APOPTOTIC BODIES
Prof Dr Turgay İSBİR

3. Cytokine-induced atropy, eg. through tumor necrosis factor
The triggering events of apoptosis is not the same in all cells. The orderly elimination of cells during apoptosis occurs in :
Development
Normal cell turnover
Cytokine-induced atropy, eg. through tumor necrosis factor
Viral induced proceeses, eg. in acquired immunomodeficency syndrome
Several neurodegenerative diseases, such as Huntington’s disease and Alzheimer disease.
Prof Dr Turgay İSBİR

4. Action of divalent cations ( Ca+2 and Zn+2 )
Several molecular events play a role initiaing apoptosis, in particular :
Action of divalent cations ( Ca+2 and Zn+2 )
Changes in the membranes of apoptotic cells
Signal transduction pathways for apoptosis.
Prof Dr Turgay İSBİR

5. DISTINCT FORMS OF PROGRAMMED CELL DEATH
There are tree distinct forms of programmed cell death :
Type 1 cell death
Type 2 cell death
Type 3 cell death
Prof Dr Turgay İSBİR

6. 1 ) TYPE 1 CELL DEATH:
Cells undergoing apoptosis shrank and display nuclear and chromatin condensation.
Externally, the cell appears to boil as the membrane becomes convoluted, an event refferred to as membrane ‘bleeding’.
The cell then fragments into membrane-bound bodies called ‘ Apoptotic bodies ’ that contain intact cellular organnelles and occasional chunk of condensed chromotion.
Apoptotic bodies are then engulfed by phagocytosis.
Prof Dr Turgay İSBİR

7. 2.) TYPE 2 CELL DEATH ( or Autophagic Cell Death ):
Type 2 cell death, another, less-studied form of programmed cell death, is characterized by the apperance of large numbers of cytoplasmic vacuoles and is associated with increased lysosomal activity.
In autophagic death death, double-membrane sheets, derived largely form the endoplasmic reticulum ( ER ) , form cytoplasmic vacuoles that engulf intracellular organelles and cytoplasmic material.
These vesicles fuse with lysosomes to become structures called Autolysomes, where the sequestered cellular components are digested. This process is independent of phagocytosis. ( Autophagy derived from Greek meaning ‘ to eat oneself’ )
Prof Dr Turgay İSBİR

8. 3.) TYPE 3 CELL DEATH:
Type 3 cell death, the least-studied type of programmed cell death, is characterized by the swelling of cellular organnelles and lysosome- independent formation of ‘empty spaces’ is the cytoplasm; it has some similuates to necrosis.
sNo chromatin condensation occurs, although chromation clusttening is sometimes seen. In contrast with apoptosis and autophagic cell death, which are observed in many types of developing animals, type 3 cell death ( or nonlysosomal cell death ) does not appear to be as common in nonpathologic conditions.
Prof Dr Turgay İSBİR

9. Prof Dr Turgay İSBİR

10. BIOLOGIC and MORPHOLOGIC FEATURES OF APOPTOSIS
 Apoptosis is a physiologic process of cell elimination in contrast to another form of cell death called NECROSIS.
Necrosis is a passive, adenosine triphosphate- independent form of cell death that requires an acute nonphysiologic injury ( i.e., ischemia, mechanical injury, and toxins ) that results in destruction of the cytoplasmic and organellar membranes with subsequent cellular swelling and lysis.
The lysis of necrotic cells releases cytoplasmic and organelle contents into the extracellular miliev, resulting in inflammation with surrounding tissue necrosis and destruction in contrast, apoptosis is a highly regulated energy-requiring form of cell death that is genetically programmed.
Prof Dr Turgay İSBİR

11. Apoptotic cells undergo the following sequenced of morphologic and biological events:
1.) Changes ın The Membranes of APOPTOTIC CELLS:
In the early phase of apoptosis, cell exhibit a shrunken cytoplasm and detach from neighbouring cells. It is important to emphassize that cells undergoing apoptosis- eg. cells in necrosis, do not pose a threat to surronding cells. Apoptotic cells are readly phagocytosed, and it appears that changes in the membrane maket he cell move ‘potable’ to the macrophage.
One feature that occurs on the membrane is a loss of sialic acids as the terminal sugar on glycoproteins and glycolipids. This seems to maket he cell more available for engulfment by the resident phagocyte it also appears that the presence of VITRONECTIN RECEPTORS on the cell’s surface attracts the macrophage. 
Other membrane changes-eg., lipid rearrengement of its phosphaditly serine to be more externally exposed-are also characteristic of some apoptotic cells.
Prof Dr Turgay İSBİR

12. 2.) Middle events include chromatin condensation with resultant cresent-shapped nuclei and nuclear fragmentation. During this phase, endonuclease activation results in the fragmentation of DNA into 180 to 200 base pair (bp) internucleosomal sized fragments.
3.) Late in apoptosis, the cells begins to fragment into plasma membrane bound vesicles termed APOPTOTIC BODIES, which are then phagocytized by neigbouring cells and macrophages without inducing an inflammatory response.
Prof Dr Turgay İSBİR

13. Signaling pathways that promote cell survival (or Apoptotic stimuli ).
The molecular machinery that apoptosis can be divided into three parts:
Signaling pathways that promote cell survival (or Apoptotic stimuli ).
Regulation by pro and antiapoptotic factors
Activation of cell’s execution (i.e; death) pathways
These molecular events results in the marphologic and biological characteristics of the apoptotic cell.
Prof Dr Turgay İSBİR

14. SIGNALING PATHWAYS THAT PROMOTE CELL SURVIVAL
 Proliferation, differentiation, and apoptosis are closely related cellular responses that appear tos hare many molecular mechanism survival factors such as neurotrophin act by suppressing the activation of caspases and other proapoptotic molecules.
In the case of neurotrophins and other related growth factors, binding to their receptors leads to receptor dimerization and autophosphorylation of receptor on spesific tyrosine residues.
Prof Dr Turgay İSBİR

15. SIGNALING PATHWAYS THAT PROMOTE CELL SURVIVAL
The cytoplasmic domain containing these phosphorylated residues serves as a dacking site for a variety of proteins, including those that regulate the activity of phosphaditylinositol 3 kinase ( PI3K ). AXT, RAF/mitogen activated protein kinase ( MEK ) extracellular signal regulated kinase ( ERK ) pathways:
Prof Dr Turgay İSBİR

16. I.) Phosphatidylinositol 3 kinase /Akt Signaling Pathways:
Activation of receptor tyrosine kinase ( RTKs ) leads to the recruitment and activation of PI3K, either directly or via adaptor proteins (such as insulin receptor substrate-1 ( IRS ).
PI3K catalyzes the phosphorylation of PI ( 4,5 ) P2 to PI (3,4,5), leading to the recruitment of Akt and 3- phosphoinositole-dependent protein kinase-1 ( PDK-1 ) to the membrane, where Akt is activated. Once activated, Akt phosphorylates and inactivates several proapoptotic protein such as GS K-3B, FOXO and BAD.
Phosphorylation of FOXO and BAD leads to their sequestration in the cytoplasm by , preventing their translocation to the nucleus and mitochondrial membrane respectively. Akt phosphorylates Ikb kinase kinase ( IKK ) and the antiapoptotic protein CREB leading to their activation.
Prof Dr Turgay İSBİR

17. I.) Phosphatidylinositol 3 kinase /Akt Signaling Pathways:
Phosphorylation of IKK results in activation of nuclear factor-kappa β leading to their activation of nuclear phosphorylation of IKK results in activation of nuclear factor-kappa β ( NF-Kβ ), a protein with strong antiapoptotic affects.
The activation of PI3K is oppesed by PTEN ( pentaerythroid tetranitrate ), a lipid phosphatase that converts PI ( 3,4,5 ) P3 back to PI ( 4,5 ) P2.
Prof Dr Turgay İSBİR

18. I.) Phosphatidylinositol 3 kinase /Akt Signaling Pathways:
Prof Dr Turgay İSBİR

19. II.) The Raf/Mitogen-Activated Protein Kinase ( MEK ) / Extracellular-Signal-Regulated Kinase ( ERK ) Pathway:
Activation of GTPase Ras occurs after stimulation of receptor tyrosine kinase ( RTKs ), a process that also involves adaptor such as Shc, Grb and Sos. Once activated ,
Ras activates Rof, which turns on MEK, which, in turn, activates ERK activation leads to the phosphorylation of procaspase-9 inhibiting its activation, inactivating it.
ERK also phosphorylate BAD, leading to its sequestration in the cytoplasm. Finally ERK activation leads to the activation of CREB, a protein with antiapoptotic affect.
Prof Dr Turgay İSBİR

20. II.) The Raf/Mitogen-Activated Protein Kinase ( MEK ) / Extracellular-Signal-Regulated Kinase ( ERK ) Pathway:
Prof Dr Turgay İSBİR

21. CASPASES
Caspases are a family of cysteine aspartat protease that represent the mammalian homologs of CED-3. Currently, 12 caspases have been identified in human ( only 10 have been identified in mice ).
Although members of this family differ in primary sequence and substrate specifity, they are all synthesized and exists normally in healthy cell as zymogens.
The procaspases are cleared to form heterodimers of two small (approximately kDa) and two large subunits (approximately kDa), which is the activate form of the enzyme.
Prof Dr Turgay İSBİR

22. Activation of caspases: Caspases are synthesized in their zymogene form requiring proteolytic processing for activation. Processing of the proenzyme at specific aspartat residues removes the N-terminal prodomain cleavage between the large and short subunits and association of two heterodimers to form a tetramer result in the generation of active caspase.
Prof Dr Turgay İSBİR

23. The sequence of Ced-3 exhibits homology to the mammalian interleucin-1β (IL-1B) converting enzyme which is currently known a caspase1. There are 10 known mammalian caspases, each of which is intimately involved in the conserved biological pathway that mediates apoptotic cell death.
Active caspase recognize a tetrapeptide sequence within substrates clearing after an aspartic acid residue at the P1 position of the sequence. For example, the preferred recognition sequence for caspase1 is Tyr-Val-Ala-Asp (Y-V-A-D), whereas the recognition sequence preferred by caspase-3 is Asp-Gk-Val-Asp (D-E-V-D
Prof Dr Turgay İSBİR

24. )Although most caspases function to regulate apoptosis, some members of the caspase family (e.g.,caspase1,-4,-5) are involved in cytokine maturation.
Caspases that regulate apoptosis have been classified into two groups referred to a “İnitiator Caspases” and “Effector Caspases”. Initiator caspases (e.g., caspases 8-9 and -10), characterized by long prodomains, have low- instrinsic-enzymatic activity even in their zymogen form.
Prof Dr Turgay İSBİR

25. “Caspase Activation by the extrinsic and instrinsic pathways”:
The extrinsic or death, receptor pathway is activated by the binding of a ligand such as Fas1, to its receptor Fas, followed by the sequential binding of the adaptor protein Fas-associated protein with death domain (FADD) and procaspase8.
Prof Dr Turgay İSBİR

26. The release of cytochromC from the mitochondria.
The formation of the death inducing signaling complex (DISC) permits the activation of procaspase8, which them proteolyzes aqnd activates caspase8.
The mitochondrial pathway also leads to the activation of caspase8, which then proteolyzes and activates caspase8.
The release of cytochromC from the mitochondria.
Prof Dr Turgay İSBİR

27. Active caspase-9 within the apoptosome can cleave caspase9.
Once in the cytoplasm, cytochromeC promotes the assembly of Apaf-1 and procaspase9 into a large complex containig seven molecules each of Apaf-1 and procaspase9.
Active caspase-9 within the apoptosome can cleave caspase9.
In addition to cytochrome C, and other proapoptotic proteins such as Smac/Diablo are released. Smac/Diablo acts by sequestening of apoptosis (IAP) away from caspases, thus permitting cell that ocur.
Prof Dr Turgay İSBİR

28. Prof Dr Turgay İSBİR

29. BCL-2 PROTEINS ( or BCL-2 Family )
The process of apoptosis is regulated by the expression of certain intracellular proteins belonging to the Bcl 2 family of genes.
In other words members of the Bcl 2 protein family are critical apoptosis regulates that act by controlling the release of cytochrome C and other apoptotic factors such as Smac/Diablo from the mitochondria Bcl 2 is a potent inhibitor of apoptosis and is predominantly expressed in chalongiocytes, colonic epithelial cells and paraeatic duct cells.
The precise mechanism of apoptotic inhibition by Bcl 2 is not known, but this protein is found on organelle membranes and may function as an antioxidant, protease inhibitor, or gate keper, preventing the apoptotic machinery from entering a target organelle.
Prof Dr Turgay İSBİR

30. BCL-2 PROTEINS
Humans have more than 20 Bcl 2 family proteins. Other proteins in this family includes Bcl-xL, Bcl-xs, Bax,Bak and Bad. Bcl-xl is another inhibitor of apoptosis. Bcl-xs, Bax, Bak and Bad function as pro-apoptotic regulators by dimenizing with Bcl 2 and Bcl-xl, inhibiting their function.
Furthermore, the pro-apoptotic Bax exhibits channel-forming activity in lipid membranes, which is blocked by Bcl 2. Increasing evidence suggests that the balance or ratio of these pro- and anti-apoptotic proteins is important for signaling in the cell to commit to or inhibit apoptosis.
Prof Dr Turgay İSBİR

31. BCL-2 PROTEINS
From function and eternal criteria, the Bcl 2 family has been divided into three groups. Members of the Group 1 family are antiapoptotic and passes all four BH domains ( BH1-4 ).
Most Group 1 Bcl 2 proteins also have a C- terminal transmembrane domain ( TMD ) with which they attack to various intracellular membranes such as the mitochondrial membrane.
Group II Bcl 2 proteins are strongly proapoptotic and lack the N-terminal BH4 domain.
Group III consist of a large group of proapoptotic that contain of BH3 domain only. Some members also support a TMD.
Prof Dr Turgay İSBİR

32. BCL-2 PROTEINS
Prof Dr Turgay İSBİR

33. THE APOPTOTIC PATHWAY OF CELL DEATH
The molecular mechanism involved in apoptosis are divided into tree parts:
FIRST :Stimuli of the apoptotic pathway include DNA damage through ionizing radiation chemotrapeutic agents ( p53 activation ) activation of death receptors such as Fas and TNF-α, free radical formation, or loss of growth factor signaling.
SECOND :The progression of the stimuli to the central execution pathway is either positevely or negatively regulated by expression of the Bcl 2 family proteins.
THIRD : The execution phase of apoptosis involves the activation of a family of evolutionarily conserved proteases called caspases. Caspase activation targets various nuclear and cytoplasmic proteins for activation or destruction leading to the morphologic and biochemical characteristics of apoptosis.
Prof Dr Turgay İSBİR

34. THE APOPTOTIC PATHWAY OF CELL DEATH
Prof Dr Turgay İSBİR