Apoptosis: MBBS, FCPS, M.Phil. Histopathology Department of Pathology CMHLMC

Apoptosis
Dr Uzma Rihan
MBBS, FCPS, M.Phil. Histopathology
Department of Pathology
CMHLMC
• Learning Objectives:
• At the end of the lecture students will be able to:
 Enlist causes of Apoptosis
 Describe mechanisms of Apoptosis
 Enlist Differences between Necrosis and Apoptosis

Apoptosis
• Apoptosis is a pathway of cell death in living tissues

that is induced by a tightly regulated suicide program in
which cells destined to die activate intrinsic enzymes
that degrade the cells' own nuclear DNA and nuclear
and cytoplasmic proteins.
• Programmed cell Death

Apoptosis
 Physiological
 Pathological
 Physiological Apoptosis:
• Death by apoptosis is a normal phenomena & it serve to to
eliminate unwanted, aged or potentially harmful cells that are no
longer needed, and to maintain a steady number of various cell
populations in tissues.
Apoptosis in Physiologic Situations
 Destruction of cells during embryogenesis;

 Implantation
 Organogenesis
 Developmental involution.
 Involution of hormone-dependent tissues upon hormone

withdrawal:
 Endometrial cell breakdown during the menstrual cycle.
 Ovarian follicular atresia in menopause.
 Regression of the lactating breast after weaning
 Testicular atrophy due to androgens deficiency.
 Destruction of cells during

embryogenesis:
 Implantation
 Organogenesis
 Developmental involution.
 Involution of hormone-dependent
tissues upon hormone
withdrawal:
 Endometrial cell breakdown during

the menstrual cycle.
 Ovarian follicular atresia in

menopause.
 Regression of the lactating breast

after weaning.
 Testicular atrophy due to

androgens deficiency.
 Cell loss in proliferating cell

populations:
 Immature lymphocytes in the bone

marrow & thymus.
 B lymphocytes in germinal centers.
 Epithelial cells in intestinal crypts,

so as to maintain a constant
number (homeostasis).
 Elimination of potentially harmful self-reactive lymphocytes.
 Death of host cells that have served their useful purpose.
 Neutrophils in an acute inflammatory response.

 Lymphocytes at the end of an immune response.
Apoptosis in Pathologic Conditions
Apoptosis eliminates cells that are injured beyond repair without
eliciting a host reaction, thus limiting surrounding tissue damage.
DNA damage:
 Radiation
 Cytotoxic anticancer drugs
 Hypoxia
• Repair mechanisms cannot cope with the injury--- triggers intrinsic

mechanisms ---- Apoptosis.
• If insult is mild by these injurious stimuli --- Apoptosis
• But larger doses of the same stimuli --Necrotic cell death

 Accumulation of Misfolded Proteins:

• Improperly folded proteins may accumulate in the cells because of:
 Mutations in the genes encoding these proteins.
 Extrinsic factors e.g. ROS.
• Excessive accumulation misfolded proteins in the ER ----ER

stress--- Apoptotic cell death.
• Examples: degenerative diseases of the CNS and other organs.

 Cell death in certain infections:

 Adenovirus, HBV, HCV and HIV infected cells die by Apoptosis.
 Cell death in tumors and in transplant rejection by Apoptosis.

 Pathologic atrophy in parenchymal organs after duct obstruction
e.g. pancreas & parotid gland by Apoptosis.
Morphological Changes in Apoptosis
 Cell shrinkage: condensation of

cytoplasm + tightly packed organelles.
 Chromatin condensation &

fragmentation of nucleus.
 Formation of cell membrane blebs.
 Fragmentation of apoptotic cells into

membrane-bound apoptotic bodies
 Phagocytosis of apoptotic bodies,

usually by macrophages.
Morphological Changes in Apoptosis
• Apoptotic cell appears as a

round or oval mass of
intensely eosinophilic
cytoplasm with fragments of
dense nuclear chromatin
MECHANISMS OF APOPTOSIS
 Activation of Caspases.
• Activation of cysteine proteases named caspases.
• More than 10 members of Caspase family.

• Functionally divided into two groups:
 Initiators------------ caspase-8 and caspase-9.

 Executioners----caspase-3 and caspase-6.
 Caspases exist as inactive pro-enzymes, or zymogens.

 Enzymatic cleavage ----------- Active.
 Active caspases----------------- Apoptosis.
MECHANISMS OF APOPTOSIS
Two phases of Apoptosis:

 Initiation phase
 Execution phase
 Pathways for initiation of apoptosis :

 Intrinsic, or mitochondrial, pathway.
 Extrinsic, or death receptor–initiated, pathway .
Initiation Phase
Intrinsic (Mitochondrial) Pathway of Apoptosis
• Increased mitochondrial membrane permeability and release of

pro-apoptotic molecules (death inducers) from mitochondrial
intermembrane space into the cytoplasm.
• The release of these mitochondrial proteins is controlled by BCL-2 (B-

cell lymphoma 2) family of proteins encoded by BCL-2 gene
• BCL-2 Family of protein,----20 members

 Anti-apoptotic proteins (BCL-2, BCL-XL,MCL-1)
 Proapoptotic proteins (BAX & BAK)
 Sensors of damage or sensors of stress (BH3-only proteins,
BAD,BID, PUMA).
 Intrinsic (Mitochondrial)
Pathway of Apoptosis
 Anti-apoptotic proteins:
 BCL2, BCL-XL and MCL-1.
• BCL-2 & BCL-XL reside in the

outer mitochondrial membranes.
• Prevent leakage of mitochondrial

proteins cytochrome c .
• Proapoptotic proteins: BAX
and BAK.
• Release of BAX, BAK when:

 Lack of survival signals
 Damaged DNA (radiations)
 Misfolded proteins induce ER
stress.
 BAX and BAK. ------ insert into

the mitochondrial membrane ----
 leakage of Cytochrome –c into
the cytoplasm.
Initiation Phase
 Into the cytosol, cytochrome c binds to a protein called APAF-1

(apoptosis-activating factor-1 --- activation of caspase-9.
 Caspase-9 cleaves adjacent caspase-9 molecules-----> Auto-

amplification process.
 Other mitochondrial proteins (Apoptosis inducing factors AIF) in the

cytoplasm bind to and neutralize inhibitors of apoptosis ( IAPs).
Initiation Phase
 Sensors of cellular damage or stress : BIM, BID, and BAD, Puma
& Noxa OR “BH3-only proteins.”
 “BH3-only proteins.” -- activate proapoptotic proteins BAX and
BAK.
 BH3-only proteins bind and block the function of BCL-2 and BCL-X
(Anti-apoptotic proteins) & decrease their synthesis.
 Net result :
 BAX-BAK activation (pro-apoptotic proteins) + loss of anti-
apoptotic BCL-2 & BCL-X ------ release of mitochondrial proteins
( cytochrom-c) in cytoplasm ------- Caspases activation
Initiation Phase
The Extrinsic Death Receptor–Pathway of Apoptosis
• Death receptors on plasma
membrane
• Cytoplasmic domain is called

the death domain.
• Best-known death receptors:
 Type 1 TNF receptor (TNFR1)

 Fas (CD95).
 Ligand for Fas is Fas ligand
(FasL).
The Extrinsic Death Receptor–Pathway of
Apoptosis
FasL is expressed on:
 T cells that recognize self

antigens.
 Cytotoxic T lymphocytes.
• When FasL binds to Fas, three

or more molecules of Fas are
brought together.
• Their cytoplasmic death

domains form a binding site
for an adapter protein FADD
(Fas-associated death
domain)
The Extrinsic Death Receptor–Pathway of Apoptosis
FADD + death receptors active
pro caspase-8.
Active Capases-8 & 10--->

activation of other pro-
caspases----> execution phase
of apoptosis.
Inhibition of Extrinsic pathway

of apoptosis:
 Protein FLIP, which bind to pro
caspases 8 and inhibit there
cleavage.
 Some viruses and normal cells
produce FLIP.
Execution Phase of Apoptosis
• Two initiating pathways:

• Mitochondrial pathway -------- Activation of caspase-9.
 Death receptor pathway ------- Activation of caspase-8 and -10.
• After activation of initiator caspases ----- activation of

executioner caspases, caspase-3 and -6.
 Activation of DNAase ----- cleavage of DNA.
 Caspases ---lysis of nuclear matrix-------fragmentation of nuclei.

Removal of Dead Cells
Apoptotic bodies : “tasty” targets for phagocytes
Changes in the membranes of Apoptotic bodies & release of “Eat me or find me’ signals ----- Migration of phagocytes.
 In healthy cells Phosphatidylserine is present on the inner leaflet of

the plasma membrane.
 In apoptotic cells this phospholipid “flips” out --- expressed on

the outer layer of the membrane------- recognized macrophages
 Some apoptotic bodies express thrombospondin, an adhesive

glycoprotein --- recognized by phagocytes.
 Macrophages produce proteins --- bind to apoptotic cells (but not

to live cells) --- target the dead cells for engulfment.
 Natural antibodies and C1q- coat the apoptotic bodies & then
recognized by phagocytes.
Disorders Associated with Dysregulated
Apoptosis
• Disorders associated with defective apoptosis and increased cell
survival.
 Low rate of apoptosis- survival of cells with DNA damage ----->

Cancer
 Defective apoptosis due to Mutation of p53 is the most common

genetic abnormality found in human cancers.
 Defective apoptosis --- failure to eliminate self-reactive lymphocytes.

 -----Autoimmune disorders.
Disorders Associated with Increased Apoptosis
and excessive cell death
 Neurodegenerative diseases;
loss of specific sets of neurons, in
which apoptosis is caused by
mutations and misfolded proteins.
 Ischemic injury : myocardial

infarction & Stroke.
 Death of virus-infected cells

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Apoptosis

• At the end of the lecture students will be able to:

 Enlist causes of Apoptosis

 Describe mechanisms of Apoptosis

 Enlist Differences between Necrosis and Apoptosis

• Apoptosis is a pathway of cell death in living tissues

• Programmed cell Death

 Destruction of cells during embryogenesis;

 Involution of hormone-dependent tissues upon hormone

 Destruction of cells during

 Endometrial cell breakdown during

 Ovarian follicular atresia in

 Regression of the lactating breast

 Testicular atrophy due to

 Cell loss in proliferating cell

 Immature lymphocytes in the bone

 B lymphocytes in germinal centers.

 Epithelial cells in intestinal crypts,

 Elimination of potentially harmful self-reactive lymphocytes.

 Death of host cells that have served their useful purpose.

 Neutrophils in an acute inflammatory response.

• Repair mechanisms cannot cope with the injury--- triggers intrinsic

• If insult is mild by these injurious stimuli --- Apoptosis

• But larger doses of the same stimuli --Necrotic cell death

 Accumulation of Misfolded Proteins:

• Excessive accumulation misfolded proteins in the ER ----ER

• Examples: degenerative diseases of the CNS and other organs.

 Cell death in certain infections:

 Cell death in tumors and in transplant rejection by Apoptosis.

 Cell shrinkage: condensation of

 Chromatin condensation &

 Formation of cell membrane blebs.

 Fragmentation of apoptotic cells into

 Phagocytosis of apoptotic bodies,

• Apoptotic cell appears as a

• More than 10 members of Caspase family.

 Initiators------------ caspase-8 and caspase-9.

 Caspases exist as inactive pro-enzymes, or zymogens.

Two phases of Apoptosis:

 Pathways for initiation of apoptosis :

• Increased mitochondrial membrane permeability and release of

• The release of these mitochondrial proteins is controlled by BCL-2 (B-

• BCL-2 Family of protein,----20 members

• BCL-2 & BCL-XL reside in the

• Prevent leakage of mitochondrial

• Release of BAX, BAK when:

 BAX and BAK. ------ insert into

 Into the cytosol, cytochrome c binds to a protein called APAF-1

 Caspase-9 cleaves adjacent caspase-9 molecules-----> Auto-

 Other mitochondrial proteins (Apoptosis inducing factors AIF) in the

• Cytoplasmic domain is called

• Best-known death receptors:

 Type 1 TNF receptor (TNFR1)

 T cells that recognize self

• When FasL binds to Fas, three

• Their cytoplasmic death

Active Capases-8 & 10--->

Inhibition of Extrinsic pathway

• Two initiating pathways: