Apoptosis / dental crown & bridge courses

JOURNAL CLUB
Apoptosis: A Review of
Programmed Cell Death
INDIAN DENTAL ACADEMY
Leader in continuing Dental Education
www.indiandentalacademy.com

REFERENCES
• Review Article- Apoptosis, Bcl-2 family
proteins and caspases. Int J Physiol
Pathophysiol Pharmacol 2009;1:97-115.
• Apoptosis and cancer: the genesis of a research
field-Nature Reviews Cancer 9, 501-507 (July
2009).
• Review article- Apoptosis: mechanisms and
clinical implications. Anaesthesia, 2000, 55,
pages 1081-1093.

REFERENCES
• Cohen, G. M., Caspases: the executioners of
apoptosis. Biochem J 326, 1–16.
• Barry, M., and Bleackley, R. C. (2002).
Cytotoxic T lymphocytes: all roads lead to
death. Nat Rev Immunol 2, 401–9.
• Ashkenazi, A., and Dixit, V. M. (1998). Death
receptors: signaling and modulation. Science
281, 1305–8.
• Robbins and Cotran: Pathologic basis of
disease.

INTRODUCTION
• Apoptosis is a pathway of cell death that is
induced by a tightly regulated suicide program
in which cells destined to die activate enzymes
that degrade the cells’ own nuclear DNA and
nuclear and cytoplasmic proteins.
• Apoptotic cells break up into fragments, called
apoptotic bodies.

• This process was recognized in 1972 by the
distinctive morphologic appearance of membrane-
bound fragments derived from cells, and named
after the Greek designation for “falling off ”.
• The process of apoptosis in mammalian cells is
transpired from the investigation of programmed
cell death that occurs during the development of
the nematode C. elegans (Horvitz, 1999).

• Apoptosis occurs normally during development
and aging and as a homeostatic mechanism to
maintain cell populations in tissues.
• Apoptosis also occurs as a defense mechanism
such as in immune reactions or when cells are
damaged by disease or noxious agents.

• Although there are a wide variety of stimuli
and conditions, both physiological and
pathological, that can trigger apoptosis, not
all cells will necessarily die in response to
the same stimulus.

• Apoptosis is a coordinated and often
energy-dependent process that involves the
activation of a group of cysteine proteases
called “caspases” and a complex cascade
of events that link the initiating stimuli to
the final demise of the cell.

MORPHOLOGY OF APOPTOSIS
• Cell shrinkage.
• Chromatin condensation.
• Formation of cytoplasmic
blebs and apoptotic bodies.
• Phagocytosis of apoptotic cells
or cell bodies, usually by
macrophages.

• Macrophages that engulf and digest apoptotic
cells are called “tingible body macrophages”
and are frequently found within the reactive
germinal centers of lymphoid follicles or
occasionally within the thymic cortex.
The tingible bodies
are the bits of nuclear
debris from the
apoptotic cells.

• On histologic examination with hematoxylin
and eosin stain, apoptosis involves single cells
or small clusters of cells.
• The apoptotic cell appears as a round or oval
mass with dark eosinophilic cytoplasm and
dense purple nuclear chromatin fragments.

DISTINGUISHING APOPTOSIS
FROM NECROSIS
• The alternative to apoptotic cell death is
necrosis, which is considered to be a toxic
process where the cell is a passive victim
and follows an energy-independent mode of
death.

COMPARISON OF MORPHOLOGICAL
FEATURES OF APOPTOSIS AND
NECROSIS
APOPTOSIS
• Single cells or small
clusters of cells.
• Cell shrinkage and
convolution.
• Pyknosis and karyorrhexis.
• Intact cell membrane.
• Cytoplasm retained in
apoptotic bodies.
• No inflammation.
NECROSIS
• Often contiguous cells.
• Cell swelling.
• Karyolysis, pyknosis and
karyorrhexis.
• Disrupted cell membrane.
• Cytoplasm released.
• Inflammation usually
present.

DISTINGUISHING APOPTOSIS
FROM NECROSIS

IS APOPTOSIS AN IRREVERSIBLE
PROCESS?
• Until recently, apoptosis has traditionally been
considered an irreversible process with caspase
activation committing a cell to death and the
engulfment genes serving the purpose of dead cell
removal.
• However, the uptake and clearance of apoptotic
cells by macrophages may involve more than just the
removal of cell debris.

• Hoeppner et al. have shown that blocking
engulfment genes in C. elegans embryos
enhances cell survival when cells are
subjected to weak pro-apoptotic signals.

• Reddien et al. demonstrated that, in C.
elegans, mutations that cause partial loss of
function of killer genes allow the survival of
some cells that are programmed to die via
apoptosis, and mutations in engulfment genes
enhance the frequency of this cell survival.

• Moreover, mutations in engulfment genes
alone allowed the survival and differentiation
of some cells that were otherwise destined to
die via apoptosis.

BIOCHEMICAL FEATURES OF
APOPTOSIS
• Activation of Caspases.
• DNA and Protein Breakdown.
• Membrane Alterations and Recognition by
Phagocytes.

Activation Of Caspases
• A specific feature of apoptosis is the activation
of several members of a family of cysteine
proteases named caspases.
• Caspases have proteolytic activity and are able
to cleave proteins at aspartic acid residues.
• Once caspases are initially activated, there
seems to be an irreversible commitment towards
cell death.

Ten major caspases have been identified and
broadly categorized into:
• Initiators (caspase-2,-8,-9,-10),
• Effectors or executioners (caspase-3,-6,-7),
• Inflammatory caspases (caspase-1,-4,-5)

• Like many proteases, caspases exist as
inactive pro-enzymes and must undergo an
enzymatic cleavage to become active.
• The presence of cleaved, active caspases is a
marker for cells undergoing apoptosis.

DNA and Protein Breakdown
• DNA breakdown by Ca2+and
Mg2+dependent endonucleases also
occurs, resulting in DNA fragments of
180 to 200 base pairs (Bortner et al.,
1995).

• A characteristic “DNA ladder” can be visualized
by agarose gel electrophoresis with an ethidium
bromide stain and ultraviolet illumination.

Membrane Alterations and Recognition
by Phagocytes
• The plasma membrane of apoptotic cells
changes in ways that promote the
recognition of the dead cells by
phagocytes.

• One of these changes is the movement of some
phospholipids (phosphatidylserine) from the
inner leaflet to the outer leaflet of the membrane,
where they are recognized by a number of
receptors on phagocytes.

• Although externalization of phosphatidylserine
is a well-known recognition ligand for
phagocytes on the surface of the apoptotic cell,
recent studies have shown that other proteins
are also be exposed on the cell surface during
apoptotic cell clearance.
• These include Annexin I and calreticulin.

• These lipids are also detectable by binding
of a protein called annexin V.
• Thus, annexin V staining is commonly used
to identify apoptotic cells.

MECHANISMS OF APOPTOSIS
• The mechanisms of apoptosis are highly
complex and sophisticated, involving an
energy-dependent cascade of molecular events.
• To date, research indicates that there are two
main apoptotic pathways: the extrinsic or
death receptor pathway and the intrinsic or
mitochondrial pathway.

• There is an additional pathway that involves T-
cell mediated cytotoxicity and perforin-
granzyme-dependent killing of the cell.
• The perforin/granzyme pathway can induce
apoptosis via either granzyme B or granzyme A.
• The extrinsic, intrinsic, and granzyme pathways
converge on the same terminal, or execution
pathway.

The Intrinsic (Mitochondrial) Pathway
of Apoptosis
• The mitochondrial pathway is the major
mechanism of apoptosis in all mammalian cells.
• This pathway of apoptosis is the result of
increased mitochondrial permeability and release
of pro-apoptotic molecules (death inducers) into
the cytoplasm.

• There is release of two main groups of normally
sequestered pro-apoptotic proteins from the
intermembrane space into the cytosol.
• The first group consists of cytochrome c, Smac,
and the serine protease HtrA2/Omi.
• These proteins activate the caspase-dependent
mitochondrial pathway.

• The second group of pro-apoptotic proteins,
AIF, endonuclease G and CAD, are released
from the mitochondria during apoptosis, but
this is a late event that occurs after the cell
has committed to die.

• The control and regulation of these apoptotic
mitochondrial events occurs through members
of the Bcl-2 family of proteins.
• The Bcl-2 family of proteins governs
mitochondrial membrane permeability and can
be either pro-apoptotic or anti apoptotic.

• To date, a total of 25 genes have been
identified in the Bcl-2 family.
• Some of the anti-apoptotic proteins include
Bcl-2, Bcl-x, Bcl-XL, Bcl-XS, Bcl-w, BAG.
• Some of the pro-apoptotic proteins include
Bcl-10, Bax, Bak, Bid, Bad, Bim, Bik, and
Blk.

• These proteins have special significance since they
can determine if the cell commits to apoptosis or
aborts the process.
• It is thought that the main mechanism of action of
the Bcl-2 family of proteins is the regulation of
cytochrome c release from the mitochondria via
alteration of mitochondrial membrane permeability.

The Extrinsic (Death Receptor–Initiated)
Pathway of Apoptosis
• This pathway is initiated by engagement of
plasma membrane death receptors on a
variety of cells.
• Death receptors are members of the TNF
receptor family that contain a cytoplasmic
domain involved in protein-protein interactions
that is called the death domain because it is
essential for delivering apoptotic signals.

• To date, the best-characterized ligands and
corresponding death receptors include
FasL/FasR, TNF-α/TNFR1, Apo3L/DR3,
Apo2L/DR4 and Apo2L/DR5.
• The sequence of events that define the extrinsic
phase of apoptosis are best characterized with
the FasL/FasR and TNF-α/TNFR1 models.

TNF Ligand
TNF Receptor
TRADD + FADD + RIP
DISC

Cytotoxic T Lymphocyte–Mediated
Apoptosis
• Cytotoxic T lymphocytes (CTLs) recognize
foreign antigens presented on the surface of
infected host cells.
• Upon activation, CTLs secrete perforin, a
transmembrane pore-forming molecule, which
promotes entry of the CTL granule serine
proteases called granzyme A and granzyme B.

• Granzyme B have the ability to cleave
proteins at aspartate residues and thus
activate a variety of cellular caspases.
• Granzyme A is also important in cytotoxic
T cell induced apoptosis and activates
caspase independent pathways.

Cytotoxic T cells
Perforin
Granzyme B Granzyme A
Execution Pathway
Caspase10
activation
Caspase3
activation

Cytotoxic T cells
Perforin
Granzyme B Granzyme A
Activates DNA nicking via
DNAse NM23-H1 SET Complex
(Tumor suppressor (nucleosome assembly protein)
gene product)
inhibits
Granzyme A protease
Cleaves SET Complex
Thus, releasing inhibition
of NM23-H1
Apoptotic DNA
degradation

Execution Pathway
• The extrinsic and intrinsic pathways both end at
the point of the execution phase, considered
the final pathway of apoptosis.
• After an initiator caspase is cleaved to generate
its active form, the enzymatic death program is
set in motion by rapid and sequential
activation of the executioner caspases.

Removal of Dead Cells
• Phagocytic uptake of apoptotic cells is the last
component of apoptosis.
• Phospholipid asymmetry and externalization
of phosphatidylserine on the surface of
apoptotic cells and their fragments is the
hallmark of this phase.

• Although the mechanism of phosphatidylserine
translocation to the outer leaflet of the cell
during apoptosis is not well understood, it has
been associated with loss of aminophospholipid
translocase activity.

• The appearance of phosphotidylserine on the
outer leaflet of apoptotic cells then facilitates
non-inflammatory phagocytic recognition,
allowing for their early uptake and disposal.
• This process of early and efficient uptake with
no release of cellular constituents, results in
essentially no inflammatory response.

APOPTOSIS IN HEALTH AND
DISEASE
Apoptosis in Physiologic Situations:
• The programmed destruction of cells during
embryogenesis, including implantation, organogenesis
& developmental involution.
• Involution of hormone-dependent tissues upon
hormone withdrawal, such as endometrial cell
breakdown during the menstrual cycle, ovarian
follicular atresia in menopause, the regression of the
lactating breast after weaning.

• Cell loss in proliferating cell populations,
such as immature lymphocytes in the bone
marrow and thymus that fail to express
useful antigen receptors, B lymphocytes in
germinal centers, and epithelial cells in
intestinal crypts, so as to maintain a constant
number (homeostasis).

• Elimination of potentially harmful self-
reactive lymphocytes, either before or after they
have completed their maturation, so as to prevent
reactions against one's own tissues.
• Death of host cells that have served their
useful purpose, such as neutrophils in an acute
inflammatory response, and lymphocytes at the
end of an immune response.

Pathologic Apoptosis
• DNA damage- Exposure of cells to radiation
or chemotherapeutic agents induces
apoptosis by a mechanism that is initiated by
DNA damage and that involves the tumor-
suppressor gene p53.
• Accumulation of misfolded proteins- in ER
leads to a condition called ER stress which
culminates in apoptotic cell death.

Disorders Associated with
Dysregulated Apoptosis
• Disorders associated with defective apoptosis
and increased cell survival- if cells that carry
mutations in p53 are subjected to DNA
damage, the cells not only fail to die but are
susceptible to the accumulation of mutations
because of defective DNA repair, and these
abnormalities can give rise to cancer.

• Disorders associated with increased apoptosis
and excessive cell death- These diseases are
characterized by a loss of cells and include-
(1) neurodegenerative diseases, manifested by loss
of specific sets of neurons, in which apoptosis is
caused by mutations and misfolded proteins.
(2) ischemic injury, as in myocardial infarction and
stroke.
(3) death of virus-infected cells, in many viral
infections. www.indiandentalacademy.com

CONCLUSION
• Apoptosis is regarded as a carefully
regulated energy dependent process,
characterized by specific morphological
and biochemical features in which
caspase activation plays a central role.

• Although many of the key apoptotic proteins
that are activated or inactivated in the
apoptotic pathways have been identified, the
molecular mechanisms of action or activation
of these proteins are not fully understood and
are the focus of continued research.

• The importance of understanding the
mechanistic machinery of apoptosis is vital
because programmed cell death is a
component of both health and disease, being
initiated by various physiologic and
pathologic stimuli.

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Apoptosis / dental crown & bridge courses

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