Programmed Cell Death

Richard A. Lockshin Mechanisms Prof.

1964 Dr. Ayhan BİLİR

2024-2025
Cell and tissue injury
Cell and tissue injury consists of a number of biochemical and morphologic changes
resulting from exogenous or endogenous causes, leading to a reversible or irreversible
disruption of normal cell function.
Exogenous causes of injury
include physical injury (trauma),
thermal injury (heat or cold),
radiation injury (ultraviolet light or
ionizing radiation),
chemical injury (caustic material),
bacterial toxicity (cholera toxin inducing
watery diarrhea),
drug toxicity (mercury toxicity to the
kidneys) and environmental injury (air
pollutants).

Endogenous causes of injury

include genetic defects (inborn errors of
metabolism) and
nutritional deficiency (intestinal
malabsorption resulting from celiac disease).
In multicellular organisms, cell death is a critical and active process that
maintains tissue homeostasis and eliminates potentially harmful cells.
There are three major types of morphologically distinct cell death:

1. Apoptosis (type I cell death),

2. Autophagic cell death (type II),
3. And necrosis (type III).

All three can be executed through distinct, and sometimes overlapping, signaling
pathways that are engaged in response to specific stimuli.

These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin

condensation, and chromosomal DNA fragmentation.
Figure 1: Classification, mechanism,
and factors involved in different key
cell death pathways Key cell death
pathways can be broadly classified
on the basis of showing systemic
death phenomenon or the presence
of apoptotic hallmarks.
Figure 2: Morphology of different key cell death pa
thways Apoptosis and anoikis are classified under
apoptotic PCD
(a). In apoptosis, nuclear DNA is fragmented, and
the membrane is blebbed, while in anoikis, the ce
lls lose their anchorage property to metastasize to
other tissues. The morphological alteration in apo
ptotic non-PCDs
Apoptosis (see 3-12 )
Under normal physiologic conditions, cells deprived of survival factors, damaged or senescent
commit suicide through an orderly regulated cell death program called apoptosis (Greek apo ,
off; ptosis , fall).

Apoptosis is different from necrosis . In a preceding section (see 3-12 ), we saw that necrosis is
a non-physiologic process that occurs after acute injury (for example, in an ischemic stroke).
Necrotic cells lyse and release cytoplasmic and nuclear contents into the environment, thus
triggering an inflammatory reaction. As was mentioned earlier, cells undergoing apoptosis lose
intercellular adhesion, fragment the chromatin and break down into small blebs called apoptotic
bodies (see 3-12 ). Apoptotic bodies are phagocytosed by macrophages, and inflammation does not
occur.
MORPHOLOGIOCAL CHANGES IN APOPTOSIS
•Cell shrinkage: Size of the cell decreases with
dense eosinophilic cytoplas
•Chromatin condensation and fragmentation–
Chromatin material aggregates below the
nuclear membrane initially. later
fragmentation of nuclear material occurs.
•Formation of cytoplasmic blebs & apoptotic
bodies – Cell surface shows blebbing.
Condensed cytoplasm, fragmented nuclear
material and organelles are packed into the
blebs which are separated from the cell
forming apoptotic bodies
•Phagocytosis of apoptotic cells or cell
bodies usually by macrophages which degrade
them by lysosomal enzymes

Histologic examination
•Tissues stained with H & E show the apoptotic
cell as round or oval mass of intensely
eosinophilic cytoplasm with dense nuclear Reference; Vinay kumar, Abul K.Abbas, Nelson Fausto, Jon C. Aster.
chromatin pattern Robbins and Cotran Pathologic basis of disease. 8th edition
•Inflammatory reaction is absent in apoptosis
Apoptotic cell death is observed during normal fetal development.
For example, the formation of fingers and toes of the fetus requires the elimination by
apoptosis of the tissue between them.
During fetal development of the central nervous system, an excess of neurons, eliminated
later by apoptosis, is required to establish appropriate connections or synapses between them

The regression of the embryonic müllerian duct in the male fetus is triggered by Sertoli
cell-derived anti-mullerian hormone, AMH ( Sperm Transport and Maturation). In the adult
female, the breakdown of the endometrium during the premenstrual phase and the
regression of the corpus luteum in the ovary are determined by a hormonal-regulated
ischemia and consequent hypoxia

Mature granulocytes in peripheral blood have a life span of 1 to 2 days before undergoing
apoptosis.
The clonal selection of T cells in the thymus (to eliminate self-reactive lymphocytes to
prevent autoimmune diseases.

Immune-Lymphatic System) and cellular immune responses involve apoptosis.

This remarkable range of functions of apoptosis arise from a precise and complex genetic and
molecular sequence.
In this worm, four genes are required for the orderly cell death program:
1. ced-3 (for cell death defective-3),
2. ced-4,
3. egl-1 (for egg laying-1) and
4. ced-9.
The products of the first three genes mediate cell death. The gene ced-9 is an inhibitor of
apoptosis.

The proteins encoded by these four genes in the worm are found in vertebrates.
Protein ced-3 is homologous to caspases;
ced-4 corresponds to Apaf-1 (for apoptotic protease activating factor-1) ,
ced-9 to Bcl-2 (for B-cell leukemia-2); and
egl-1 is homologous to Bcl-2 homology region 3 (BH3)-only proteins.

Extrinsic signals bind to cell surface receptors (for example, Fas ligand and granzyme
B/perforin).
Intrinsic signals (for example, the release of cytochrome c from mitochondria) can trigger cell
death.
Caspases can be upstream initiators and downstream executioners of cell death.

Upstream (initiator) procaspases include

procaspases 8, 9 and 10 with a long N-terminal prodomain called CARD (for caspase-
recruiting domain) .

Downstream (executioner) procaspases comprise procaspases 3, 6 and 7 with

a short N-terminal prodomain called DED (for death-effector domain) .

Caspase Activation, Function, and Regulation

Apoptosis involves the activation of caspases, which orchestrate all of the morphological
changes that characterize this form of cell death. Caspases are cysteine proteases with
specificity for aspartic acid residues in their substrates. Although at least 17 different caspases
exist in mammals, our focus is on only a subset of these for which the activation is at least
partially understood and roles in cell death have been established.
Intrinsic pathway: Mitochondria cytochrome c

Mitochondria are central to the intrinsic apoptosis pathway.

Cytochrome-c is a component of the mitochondria electron-transporting chain
involved in the production of ATP and also a trigger of the caspase cascade.

The cell death pathway can be activated when cytochrome c is released from the
mitochondria into the cytoplasm. It is thought that during apoptosis, mitochondria
DNA (mtDNA) is also released into the cytoplasm.
Bcl-2 family members can have proapoptotic or antiapoptotic activities. Bcl-2 and
Bcl-xL have antiapoptotic activity. Bax, Bak, Bid and Bad are proapoptotic
proteins .
Apoptosis is mediated by the proapoptotic proteins Bax and Bak
APOPTOSIS AND THE IMMUNE SYSTEM
ALPS is characterized by the accumulation of mature lymphocytes in lymph nodes and spleen
causing lymphoadenopathy (enlargement of lymph nodes)
and splenomegaly (enlargement of the spleen).

APOPTOSIS AND NEURODEGENERATIVE DISEASES

Neurologic diseases are examples of the mechanism of cell death.
For example, an ischemic stroke can cause an acute neurologic disease in which necrosis
and activation of caspase 1 are observed.

Necrotic cell death occurs in the center of the infarction, where the damage is severe.
Apoptosis may be observed at the periphery of the infarction, because the damage is
not severe due to collateral blood circulation. Pharmacologic treatment with caspase inhibitors
can reduce tissue damage, leading to neurologic improvement.
Apoptosis is triggered when cell-surface death receptors such as Fas are bound by their
ligands (the extrinsic pathway) or when Bcl2-family proapoptotic proteins cause the
permeabilization of the mitochondrial outer membrane (the intrinsic pathway).
Both pathways converge on the activation of the caspase protease family, which is ultimately
responsible for the dismantling of the cell.
Detecting DNA Fragmentation.
Many of the assays used to detect apoptosis analyze the characteristic DNA fragmentation that
occurs during apoptosis.
In apoptotic cells, the genomic DNA is cleaved to multimers of 180–200bp (based on
the nucleosomal repeat length).
This cleaved DNA is easily observed as a “ladder” upon analysis by gel electrophoresis. To detect this
DNA fragmentation at the single-cell level, assays rely on labeling the ends of the nucleosomal
fragments followed by either colorimetric or fluorescent detection. This approach, commonly called
TUNEL (TdT-mediated dUTP Nick End Labeling), relies on labeling permeabilized cells to label
the 3´-OH ends of the fragments with either a fluorescent or biotinylated dUTP.
Necrosis can be recognized by specific microscopic and macroscopic
changes.
Microscopically, in addition to cell membrane breakdown caused by cell swelling,
1. Coagulative necrosis , the most common form of necrosis resulting from vascular occlusion, is
characterized by a paler than normal tissue area that retains its overall shape yet all cell functions
have stopped.
2. Liquefactive necrosis is recognized by the softening of the necrotic tissue c
aused by hydrolytic lysosome enzymes released from dead cells and neutrophils.
Examples include brain infarct ,
3. The crumble consistency and opaque aspect of the necrotic tissue in caseous necrosis , found
tuberculous and histoplasmosis granulomas (nodular inflammatory lesions), mimics cottage
cheese.
4. Fat necrosis occurs after enzymatic and traumatic injury.
Enzymatic fat necrosis involves adipose tissue within and around the pancreas.
5. Fibrinoid necrosis is restricted to the smooth muscle wall of small arteries, arterioles and
renal glomeruli affected by autoimmune diseases such as systemic lupus erythematosus.
Regulation of anoikis by extrinsic death
receptor pathways

Cell fate of normal cells and metastatic tumor

cells after losing adhesion to the extracellular
matrix.
When normal cells lose their adhesion to the
extracellular matrix, apoptosis occurs because
the body prevents the wrong connection of cells.
However, after losing adhesion to the
extracellular matrix due to its metastasis
characteristics, tumor cells develop resistance,
survive without adhesion to the extracellular
matrix, and can flow to other tissues and organs
through blood and lymph for adhesion and
metastasis
Different death receptors bind to different ligands to activate downstream caspases.
Anoikis is affected by various pathways [10] and reportedly causes cell death due to loss of cell adhesion and
ECM separation [11]. Anoikis, necrosis, ferroptosis, pyroptosis, and apoptosis are different forms of cell
death

Necrosis is an irregular and passive form of cell death, typically caused by external factors such as trauma,
infection, or ischemia. Necrosis is characterized by cell membrane rupture and leakage of cellular contents,
without the requirement of caspase proteinase activation.

Ferroptosis typically occurs due to the excessive accumulation of intracellular iron ions, leading to increased
oxidative stress within the cell. This form of cell death involves aberrant regulation of iron ion transport,
storage, and metabolism, resulting in oxidative damage and cell death.

Pyroptosis is an inflammatory form of cell death, usually occurring in response to infection or abnormal
intracellular signaling. It is characterized by cell membrane rupture and release of cellular contents,
accompanied by the activation of intracellular inflammatory factors and the occurrence of inflammatory
reactions. The signaling pathway of pyroptosis involves the formation of inflammasomes and the activation
of inflammatory-related proteinase caspase-

Apoptosis is a highly regulated and active form of cell death, involving both intrinsic and extrinsic pathways.
The intrinsic pathway primarily involves the release of mitochondria and the activation of caspases, while the
extrinsic pathway mainly involves the activation of cell membrane receptors and caspases.
 Autophagic Cell Death
(type II),
Autophagy is a highly conserved
eukaryotic cellular recycling process.
Through the degradation of cytoplasmic
organelles,
proteins,
and macromolecules,
and the recycling of the breakdown
products,
autophagy plays important roles in cell
survival and maintenance.

In mammalian cells, there are three

primary types of autophagy:
microautophagy, macroautophagy, and
chaperone-mediated autophagy (CMA).
DU-145 Prostat Cander Cell Culture(Autophagic Vacuoles)
Mechanism
A critical regulator of autophagy induction is the kinase mTOR, which when activated,
suppresses autophagy and when not activated promotes it.
Currently, 32 different autophagy-related genes (Atg) have been identified by genetic screening in
yeast

The ATG genes control the autophagosome formation through ATG12-ATG5 and LC3-II (ATG8-II)
complexes.
ATG12 is conjugated to ATG5 in a ubiquitin-like reaction that requires ATG7 and ATG10.
The Atg12–Atg5 conjugate then interacts non-covalently with ATG16 to form a large complex.

LC3/ATG8 is cleaved at its C terminus by ATG4 protease to generate the cytosolic LC3-I. LC3-I is
conjugated to phosphatidylethanolamine (PE) also in a ubiquitin-like reaction that requires Atg7
and Atg3.
The lipidated form of LC3, known as LC3-II, is attached to the autophagosome membrane.
AUTOPHAGY
Isc 10mM 72s 8X10k.jpg Isc 24s 50mM 4x10K.jpg
No.20662 No:20658
Abnormal Programmed Cell Death in Disease
Programmed cell death pathway dysfunction can lead to a range
of diseases.14

•Neurodegenerative diseases
•Cancer
•Developmental disorders
•Immunodeficiency
•Autoimmune diseases
Programmed Cell Death in Neurodegenerative Diseases
Programmed cell death dysregulation can be dangerous for disease initiation and progression.
An example of this is in neurodegenerative diseases such as Parkinson’s disease, Huntington’s
disease, and amyotrophic lateral sclerosis (ALS).15
In ALS, there is aberrant motor neuron death in the spinal cord and cerebral cortex, leading to
muscle weakness. Over 20 percent of patients with ALS have a mutation in a protein called
superoxide dismutase 1 (SOD1).
This mutation causes SOD1 to bind and block an anti-apoptotic protein, B cell lymphoma-2
(BCL-2), in spinal cord neurons, which promotes aberrant apoptosis.
What did we learn?
Thank you for your listening