An important basic and advanced course of life science

Cellular and
Molecular Biology:
Concepts and
Experiments

Department of Biotechnology
Zhi Huang (黄峙), Ph.D & Prof.
Email: thsh@jnu.edu.cn, Ph: 13678988403, qq:1547461148
 Chapter 11 Apoptosis

Learning Objectives:
1. Cell death: necrosis and apoptosis
2. Apoptotic Pathways
3. Apoptosis Assay Methods
11.1 Cell death: necrosis and apoptosis

 Cell death can occur by either of two distinct

mechanisms: necrosis or apoptosis.
 Necrosis (“accidental” cell death) is the
pathological process which occurs when cells are
exposed to a serious physical or chemical insult.
 Apoptosis (“normal” or “programmed” cell
death) is the physiological process by which
unwanted or useless cells are eliminated during
development and other normal biological
processes.
 Cytotoxicity is the cell-killing property of a
chemical compound (such as a food, cosmetic, or
pharmaceutical) or a mediator cell (cytotoxic T
cell). In contrast to necrosis and apoptosis, the term
cytotoxicity does not indicate a specific cellular
death mechanism.
 Cell-mediated cytotoxicity (that is, cell death
mediated by either cytotoxic T lymphocytes [CTL] or
natural killer [NK] cells) combines some aspects of
both necrosis and apoptosis.
 Morphological features of necrosis and apoptosis:
Differences between necrosis and apoptosis
 There are many observable morphological and biochemical
differences between necrosis and apoptosis.
Features of necrosis
• Necrosis occurs when cells are exposed to abnormal
physiological conditions: extreme variance (e.g.,
hypothermia, hypoxia) which may result in damage to the
plasma membrane, and agents (e.g., complement and lytic
viruses) which directly evoke damage to the plasma
membrane under physiological conditions.
• Necrosis begins with an impairment of the cell’s ability to
maintain homeostasis, leading to an influx of water and
extracellular ions. Organelles, most notably the mitochondria,
and the entire cell swell and rupture (cell lysis).
• Due to the ultimate breakdown of the plasma membrane, the
cytoplasmic contents including lysosomal enzymes are
released into the extracellular fluid. Therefore, in vivo,
necrotic cell death is often associated with extensive tissue
damage resulting in an intense inflammatory response.
Features of apoptosis
• Apoptosis, in contrast, is a mode of cell death that occurs
under normal physiological conditions and the cell is an
active participant in its own demise (“cellular suicide”).
• It plays key roles and is most often found during normal cell
turnover and tissue homeostasis, embryogenesis, induction
and maintenance of immune tolerance, development of the
nervous system and endocrine-dependent tissue atrophy.
• Cells undergoing apoptosis show characteristic
morphological and biochemical features, overall shrinkage in
volume of the cell and its nucleus. These features include
chromatin aggregation, nuclear and cytoplasmic
condensation, partition of cytoplasm and nucleus into
membrane bound-vesicles (apoptotic bodies) which contain
ribosomes, morphologically intact mitochondria and nuclear
material.
Features of apoptosis
• In vivo, these apoptotic bodies are rapidly recognized and
phagocytized by either macrophages or adjacent epithelial
cells. Due to this efficient mechanism for the removal of
apoptotic cells in vivo no inflammatory response is elicited.
• In vitro, the apoptotic bodies as well as the remaining cell
fragments ultimately swell and finally lysis. This terminal
phase of in vitro cell death has been termed “secondary
necrosis”.
 A comparison of normal and apoptotic cells

Scanning EM of a normal (a) and

apoptotic (b) T-cell hybridoma.
The apoptotic cell exhibits many
surface blebs that are budded off
in the cell. (c) Transmission EM
of an apoptotic cell treated with
an inhibitor that arrests apoptosis
at the membrane blebbing stage.
Apoptosis can be divided into two phase:
• An activation phase, when the cell responds to “death
signals” that commit it to undergoing self-destruction.
• An execution phase, when the death sentence is carried
out.
• Death by apoptosis is a neat, orderly process.
11.2 Apoptotic Pathways

The receptor mediated pathway of apoptosis: the

best understood apoptotic pathway activated by
stimuli that originate in the extracellular
environment is carried out by a protein called
tumor necrosis factor (TNF), which was named for
its ability to kill tumor cells.
The mitochondria mediated pathway of apoptosis:
internal stimuli, such as irreparable DNA damage,
extremely high concentration of cytosolic Ca2+, or
severe oxidative stress lead to the activation of
proapoptotic cytoplasmic factors, of which Bcl-2
family of proteins are best studied.
The receptor mediated pathway
of apoptosis. When TNF binds to a
TNF receptor (TNFR1), the activated
receptor binds two different
cytoplasmic adaptor proteins
(TRADD and FADD) and
procaspase-8 to form a multiprotein
complex at the inner surface of the
plasma membrane. The cytoplasmic
domains that are present in each
protein (green). Procaspase-8 and
FADD interact by means of
homologous regions call death
effector domains (brown). Once
assembled in the complex, the two
procaspase molecules cleave one
another to grnerate an active
caspase 8, which cleave
downstream caspases that carry out
the death sentence.
The mitochondria mediated
pathway of apoptosis. Various types
of cellular stress cause proapoptotic
members of the Bcl-2 family of
proteins, such as Bad or Bax, to
become inserted into the outer
mitochondrial membrane. Insertion of
these proteins leads, by an unidentified
mechanism, to the release of
cytochrome C molecules from the
intermembrane space of the
mitochondria. Once in the cytosol, the
cytochrome C molecules form a
multisubunit complex with a cytosolic
protein called Apaf-1 and procaspase-9
molecules. Procaspase-9 molecules
are apparently activated to their full
proteolytic capacity as the result of a
conformational change induced by
association with Apaf-1.
Key elements of the apoptotic pathway:
• Death receptors: Apoptosis has been found to be induced
via the stimulation of several different cell surface receptors in
association with caspase activation. For example, the CD95
(APO-1,Fas) receptor ligand system is a critical mediator of
several physiological and pathophysiological processes,
including homeostasis of the peripheral lymphoid
compartment and CTL-mediated target cell killing. Upon
cross-linking by ligand or agonist antibody, the Fas receptor
initiates a signal transduction cascade which leads to
caspase-dependent programmed cell death.
• Membrane alterations: In the early stages of apoptosis,
changes occur at the cell surface and plasma membrane.
One of these plasma membrane alterations is the
translocation of phosphatidylserine (PS) from the inner side
of the plasma membrane to the outer layer, by which PS
becomes exposed at the external surface of the cell.
• Protease cascade: Signals leading to the activation of a
family of intracellular cysteine proteases, the caspases,
(Cysteinyl-aspartate-specific proteinases) play a pivotal role in
the initiation and execution of apoptosis induced by various
stimuli. At least 11 different members of caspases in
mammalian cells have been identified. Among the best-
characterized caspases is caspase-1 or ICE (Interleukin-1β-
Converting Enzyme), which was originally identified as a
cysteine protease responsible for the processing of
nterleukin1β.
• Mitochondrial changes: Mitochondrial physiology is
disrupted in cells undergoing either apoptosis or necrosis.
During apoptosis mitochondrial permeability is altered and
apoptosis specific protease activators are released from
mitochondria. Specifically, the discontinuity of the outer
mitochondrial membrane results in the redistribution of
cytochrome C to the cytosol followed by subsequent
depolarization of the inner mitochondrial membrane.
• DNA fragmentation: The biochemical hallmark of apoptosis
is the fragmentation of the genomic DNA, an irreversible
event that commits the cell to die and occurs before changes
in plasma membrane permeability (prelytic DNA
fragmentation). In many systems, this DNA fragmentation
has been shown to result from activation of an endogenous
Ca2+ and Mg2+-dependent nuclear endonuclease. This
enzyme selectively cleaves DNA at sites located between
nucleosomal units (linker DNA) generating mono- and
oligonucleosomal DNA fragments.
11.3 Apoptosis Assay Methods

 Methods for studying apoptosis in cell populations.

 The DNA fragments may be assayed in either of two ways:
• Apoptotic DNA Ladder assay
• Quantification of histone complexed DNA fragments
with an ELISA method
 Caspase activation can be analyzed in different ways:
• In vitro enzyme assay. Activity of a specific caspase, for
instance caspase 3, can be determined in cellular lysates by
capturing of the caspase and measuring proteolytic cleavage
of a suitable substrate.
• Detection of cleavage of an in vivo caspase substrate. For
instance caspase 3 is activated during early stages. Its
substrate PARP (Poly-ADP-Ribose-Polymerase) and the
cleaved fragments can be detected by westernblotting.
The biochemistry of DNA fragmentation and the
appearance of DNA ladder.
Compartmentalization of HMW and LMW DNA in
normal and apoptotic cells.
Detection of cleaved PARP in cell
extracts of apoptotic cells.
 Methods for studying
apoptosis in individual cells.
 The TUNEL enzymatic labeling assay: Comparison of direct
and indirect labeling of DNA strand breaks in apoptotic cells