Programmed cell death, or apoptosis, is a tightly regulated suicide program in cells. It involves the activation of enzymes that degrade a cell's own DNA and proteins. Apoptotic cells form membrane-bound fragments that are quickly cleared by phagocytes without causing inflammation. Apoptosis occurs normally during development and aging to eliminate unwanted cells, and is also triggered when cells are damaged beyond repair. The main pathways of apoptosis are the intrinsic mitochondrial pathway initiated by cellular stress, and the extrinsic death receptor pathway initiated by signals like Fas ligand. Both pathways activate caspases that degrade cellular components and lead to the characteristic morphologic changes of apoptosis.

1. Apoptosis
Programmed cell death
“Falling off“

2. • pathway of cell death that is induced by a tightly
regulated suicide program in which cells activate
enzymes that degrade the cells' own nuclear DNA
and nuclear and cytoplasmic proteins
• Apoptotic cells  apoptotic bodies-- contain
portions of the cytoplasm and nucleus
• The plasma membrane  intact but structure is
altered  “tasty” targets for phagocytes.
• dead cell fragments ---devoured, before the
contents leaked out does not elicit an
inflammatory reaction

3. CAUSES OF APOPTOSIS
occurs normally both during development and
throughout adulthood, and serves to eliminate
unwanted, aged or potentially harmful cells.
pathologic event when diseased cells become
damaged beyond repair and are eliminated

4. Apoptosis in Physiologic Situations
• embryogenesis, including implantation, organogenesis,
developmental involution, and metamorphosis
• Involution ---- hormone withdrawal
• endometrial cell breakdown -- menstrual cycle
• ovarian follicular atresia in menopause
• the regression of the lactating breast after weaning
• prostatic atrophy after castration
• Cell loss in proliferating cell populations--- immature
lymphocytes in the bone marrow and thymus, B
lymphocytes in germinal centers, and epithelial cells in
intestinal crypts---- to maintain a constant number
• harmful self-reactive lymphocytes
• neutrophils post acute inflammatory response, and
lymphocytes at the end of an immune response----deprived
of necessary survival signals, such as growth factors.

5. Apoptosis in Pathologic Conditions
injured beyond repair
• DNA damage. Radiation, cytotoxic anticancer drugs, and
hypoxia can damage
• Accumulation of misfolded proteins----mutations / free
radicals- ER stress
• certain infections--- viral infections--- as in adenovirus and
HIV infections) or by the host immune response
• Pathologic atrophy in parenchymal organs after duct
obstruction---- pancreas, parotid gland, and kidney.

6. MORPHOLOGIC CHANGES IN APOPTOSIS

7. • Cell shrinkage---- smaller in size; the cytoplasm is
dense ---organelles--- tightly packed
• Chromatin condensation. --- peripherally, under
the nuclear membrane, into dense masses of
various shapes and sizes  nucleus may break up
into fragments.
• Cytoplasmic blebs and apoptotic bodies --
extensive surface blebbing membrane-bound
apoptotic bodies
• Phagocytosis --- by macrophages.
• Plasma membranes --- remain intact until the last
stages
• hematoxylin and eosin----- round or oval mass of
intensely eosinophilic cytoplasm with fragments of
dense nuclear chromatin -- not elicit inflammation

9. Biochemical Features of Apoptosis

10. Activation of Caspases
• activation of a family of cysteine proteases ---
caspases
• “c” ----- cysteine protease
• “aspase” ----unique ability of these enzymes to
cleave after aspartic acid residues
• caspase > 10 members, two groups—initiator and
executioner
• Initiator -- caspase-8 and caspase-9
• Executioners-- caspase-3 and caspase-6
• inactive pro-enzymes, or zymogens

11. DNA and Protein Breakdown
• characteristic breakdown -- large 50- to 300-
kilobase pieces
• Subsequent cleavage of DNA by Ca2+- and Mg2+-
dependent endonucleases -- 180 to 200 base pairs
• electrophoresis --- “ladders”
• “smeared” pattern --- necrosis

13. Membrane Alterations and Recognition by
Phagocytes
movement of phospholipids (notably
phosphatidylserine) from the inner leaflet to the
outer leaflet of the membrane
also detectable by binding of a protein --- annexin V

14. MECHANISMS OF APOPTOSIS
• imbalance in death or survival signals
• basic mechanisms of apoptosis—the genes and
proteins that control the process and the sequence
of events—are conserved in all multicellular
organisms

15. divided into
• initiation phase----some caspases become
catalytically active
• execution phase---- other caspases trigger the
degradation of critical cellular components
• Initiation
• the intrinsic, or mitochondrial, pathway
• extrinsic, or death receptor–initiated, pathway

17. The Intrinsic (Mitochondrial)
Pathway of Apoptosis
• major mechanism
• result of increased mitochondrial permeability and
release of pro-apoptotic molecules (death inducers)
into the cytoplasm
• Mitochondria ----- cytochrome c --- essential for life,
but when released into cytoplasm (an indication that
the cell is not healthy), initiate --- apoptosis
• release of mitochondrial proteins -- controlled by ----
balance between pro- and anti-apoptotic members of
the Bcl family
• named after Bcl-2, oncogene in B-cell lymphoma --
homologous to C. elegans protein Ced-9
• > 20 members of the Bcl family --regulate apoptosis.

18. • Growth factors / survival signals  anti-apoptotic ---- Bcl-2, Bcl-XL, and
Mcl-1 ---normally reside in cytoplasm , ER and mitochondrial
membranes -- control mitochondrial permeability and prevent leakage
of mitochondrial proteins
• deprived of survival signals / DNA is damage/ misfolded proteins 
sensors of damage or stress are activated
• sensors/ arbitors --- members of the Bcl family ---- Bim, Bid, Bad ,
Puma, noxa
• sensors --- activate two critical (pro-apoptotic) effectors--- Bax and
Bak, bac, p53---- form oligomers that insert into the mitochondrial
membrane --- create channels to allow proteins from the inner
mitochondrial membrane to leak out into the cytoplasm
• BH3--- bind to and block the function of Bcl-2 and Bcl-x
• mitochondrial proteins -- activate the caspase cascade -- cytochrome c
• cytochrome c binds --- Apaf-1 (apoptosis-activating factor-1,
homologous to Ced-4 in C. elegans)----forms a wheel-like hexamer 
apoptosome.
• This complex --- bind caspase-9---critical initiator caspase ----- auto-
amplification process
• Other mitochondrial proteins ---- Smac/DIABLO, enter the cytoplasm,
where they bind to and neutralize cytoplasmic proteins that function as
physiologic inhibitors of apoptosis (called IAPs that block the activation
executioners like caspase-3

21. The Extrinsic (Death Receptor–Initiated) Pathway of Apoptosis
• initiated by ---- plasma membrane death receptors
• Death receptors --- members of the TNF receptor family ----contain a
cytoplasmic domain involved in protein-protein interactions that is
called the death domain
• type 1 TNF receptor (TNFR1) ---- called Fas (CD95)
• ligand --- Fas ligand (FasL)----expressed on T cells that recognize self
antigens ( eliminate self-reactive lymphocytes), and cytotoxic T
lymphocytes (which kill virus-infected and tumor cells)
• FasL binds to Fas---- >3 molecules of Fas are brought together ---
cytoplasmic death domains--- binding site for an adapter protein that
also contains a death domain called FADD (Fas-associated death
domain)
• FADD --- in turn binds ---caspase-8 (and, in humans, caspase-10), again
via a death domain
• Multiple pro-caspase-8--- generate active caspase-8.
• enzyme then triggers a cascade of other pro-caspases ---- active
enzymes of execution phase of apoptosis
• Inhibitor -- FLIP

23. • extrinsic and intrinsic pathways --- distinct
because they involve fundamentally different
molecules for their initiation
• but there may be interconnections between them

24. The Execution Phase
• initiating pathways converge --- cascade of caspase
activation --- mediates the final phase of apoptosis
• caspase-3 and -6 --- act on many cellular
components.
• cleave an inhibitor of a cytoplasmic DNase
• degrade structural components of the nuclear
matrix---- fragmentation of nuclei

25. Removal of Dead Cells
• apoptotic bodies breaks cells --- fragments edible for
phagocytes
• phagocytosed--- before they undergo secondary
necrosis and release their cellular contents (injurious
inflammation)
 Phosphatidylserine a phospholipid “flips” out on the
outer layer of the membrane --- recognized by several
macrophage receptors
 Cells --- secrete soluble factors that recruit phagocytes
Thrombospondin, a glycoprotein ---recognized by
phagocytes
Apoptotic bodies -- coated with natural antibodies and
complement system, notably C1q---- recognized by
phagocytes

26. CLINICO-PATHOLOGIC CORRELATIONS:
APOPTOSIS IN HEALTH AND DISEASE

27. Examples of Apoptosis
1. Growth Factor Deprivation
Hormone-sensitive cells deprived of the relevant
hormone, lymphocytes that are not stimulated by
antigens and cytokines, and neurons deprived of
nerve growth factor die by apoptosis
 apoptosis is triggered by the intrinsic
(mitochondrial) pathway

28. 2. DNA Damage
• radiation or chemotherapeutic agents -- genotoxic stress----
involves the tumor-suppressor gene p53
• p53 protein accumulates –if DNA is damaged --- arrests the
cell cycle for repair
• if the damage is too great ---- p53 triggers apoptosis
• p53 mutated or absent --- mutations --neoplastic
transformation
3. Protein Misfolding.
• Chaperones --- control the proper folding of proteins
• misfolded --- proteolysed
• If misfolded proteins accumulate ---- trigger --- unfolded
protein response
• if this response is unable to cope --- activates caspases and
induces apoptosis --ER stress

30. 4. Apoptosis Induced By the TNF Receptor Family.
• FasL on T cells binds to Fas on the same or
neighboring lymphocytes
• This interaction --- elimination of lymphocytes that
recognize self-antigens
• mutations affecting Fas or FasL --- autoimmune
diseases in humans

31. 5. Cytotoxic T Lymphocyte–Mediated Apoptosis.
• CTLs recognize foreign antigens on infected host
cells
• Upon activation ---secrete perforin --- promotes
entry of serine proteases called granzymes
• Granzymes --- cleave proteins -- activate a variety
of cellular caspases --- kills target cells by directly
inducing the effector phase
Dysregulated Apoptosis

38. THANK YOU
John E. Sulston won the Nobel Prize in
Medicine in 2002, for his pioneering
research on apoptosis