CHAPTER 46

Programmed Cell Death

Necessity for Cell Death in stimuli, particularly when induction of apoptosis is

Multicellular Organisms
The ability to undergo programmed cell death (Box
46.1) is a built-in latent capacity in most cells of multi-
cellular organisms. Cell death is important for embryonic
development, maintenance of tissue homeostasis, estab-
lishment of immune self-tolerance, killing by immune
effector cells, and regulation of cell viability by hormones
and growth factors. Both extrinsic signals and internal
imbalances can lead cells to kill themselves. Furthermore,
many metazoan cells will die if they fail to receive sur-
vival signals from other cells. Abnormalities of the cell
death program contribute to several important diseases,
including cancer, Alzheimer disease, and AIDS. Cell
death programs are ancient: much of the current network
was present in the last eumetazoan common ancestor
(see Fig. 2.8).
inhibited. Programmed necrosis looks morphologically
like accidental cell death. A third pathway leading to cell
death involves autophagy (see Fig. 23.7). Although
usually regarded as a protective response to starvation,
autophagy has been implicated in certain examples of
cell death, particularly during development.
Necrosis corresponds to what most of us naively
imagine cell death would be like. Owing to lack of cel-
lular homeostasis, water rushes into the dying cell,
causing it to swell until the plasma and organelle mem-
branes burst. As a result, the cell undergoes a generalized
NEIGHBORING CELL
Fas ligand
TNF
Fas receptor
TNF
receptor

Programmed Cell Death Versus Accidental Extrinsic

DISC
Cell Death: Apoptosis Versus Necrosis pathway
Complex II Complex I
Active
Although cells die in many ways, it is useful to focus on Bcl2 caspases
killer
two poles of this spectrum: apoptosis and necrosis. Fig. Necroptosis
Survival

46.1 summarizes the major pathways of programmed

Apoptosis Inflammation
cell death. (Details are filled in as we progress through
the chapter.) Apoptosis, the most widely studied MITOCHONDRION Pyroptosis
pathway for programmed cell death, is cellular suicide Apoptosome
resulting from activation of a dedicated intracellular Bcl2
killer
Signaling
Inflammasome
program. Fig. 46.2 shows a detailed description of the Intrinsic platforms
pathway
events of apoptosis. At the other end of the spectrum Pathogen
DNA damage, etc
necrosis, also called accidental cell death, occurs when attack NUCLEUS
cells sustain a structural or chemical insult that causes
FIGURE 46.1 OVERVIEW OF PROGRAMMED CELL DEATH
the cells to swell and undergo membrane lysis (Fig.
DISCUSSED IN THIS CHAPTER. All these types of death hinge on
46.3). Examples of such insults include extremes of the assembly of a signaling platform (boxed) that is often involved in
temperature and physical trauma. Cells can also initiate activating proteases called caspases. These pathways are all dis-
active programmed necrosis in response to certain cussed in greater detail in the text.

797
798 SECTION X n Cell Cycle

BOX 46.1 Key Terms Apoptosis

Apoptosis: Type of programmed cell death that was

identified due to a particular pattern of morphologic Junctions
changes but now is defined by the action of molecular Mitochondria
pathways involving cell surface receptors or mitochon-
dria and resulting in the activation of specialized pro- Nucleus
teases. The name comes from the Greek, referring to
shedding of the petals from flowers or leaves from
Microvilli contract
trees. Apoptotic death occurs in two phases. During Intercellular junctions break
the latent phase, the cell looks morphologically normal Chromatin begins to condense
but is committed to death. The execution phase is
characterized by a series of dramatic structural and
biochemical changes that culminate in fragmentation
of the cell into membrane-enclosed apoptotic bodies.
Activities that cause cells to undergo apoptosis are said
to be proapoptotic. Activities that protect cells from
apoptosis are said to be antiapoptotic.
Autophagic Cell Death: It is still debated how widely Cell shrinks
autophagy is used as a pathway for cell death, although Chromatin condenses around
it is accepted that the pathway (which is widely nuclear periphery
assumed to be primarily a survival pathway when cells
are starved for nutrients) can promote cell death during
development. Autophagy may also either promote or
inhibit apoptosis under specialized circumstances.
Necroptosis: Programmed necrosis that occurs when
tumor necrosis factor (TNF) and certain other cell-
surface receptors are activated. Activation of these
receptors normally leads to a proinflammatory response
and cell survival, but can lead to apoptosis. If certain Cell blebs violently
Chromatin condensation
components of the apoptotic pathway are missing, continues
cells instead undergo necroptosis, apparently as a
backup pathway.
Necrosis (Accidental Cell Death): Death that results
from irreversible injury to the cell. Cell membranes
swell and become permeable. Lytic enzymes destroy
the cellular contents, which then leak out into the inter-
cellular space, leading to an inflammatory response.
Programmed Cell Death: Any active cellular process
that culminates in cell death. This may occur in Cell fragments into membrane-
response to developmental or environmental cues or enclosed apoptotic bodies
as a response to physiological damage detected by the
cells internal surveillance networks.
Pyroptosis: Often in response to intracellular pathogens,
this involves activation of caspase 1. The infected
cells secrete interleukin-1 and interleukin-18, which
promote an inflammatory response, and also undergo
a form of cell death that resembles necrosis.

Apoptotic bodies phagocytosed

by neighboring cells and
roving macrophages

FIGURE 46.2 APOPTOSISACTIVE SUICIDETYPICALLY

AFFECTS SINGLE CELLS. Neighboring cells remain healthy. Apop-
totic cell death usually does not lead to an inflammatory response.
 CHAPTER 46 n Programmed Cell Death 799

Necrosis
Trauma Dissolution of
cellular structures

Junctions

Mitochondria

Nucleus

Cells and organelles swell Cell lysis

Chromatin condenses Invasion of phagocytic cells
Membranes compromised: Inflammation
H 2O fluid rushes in H2O

FIGURE 46.3 NECROSIS USUALLY RESULTS FROM IRREVERSIBLE INJURY TO CELLS. Typically, groups of cells are affected. In most
cases, necrotic cell death leads to an inflammatory response (red activated macrophages).

Point of Reprieve
Insult no return (very rare)

Latent Execution
Condemned Committed
Rescue by Rescue usually Morphologic
survival factors impossible changes
FIGURE 46.4 TWO PHASES OF APOPTOSIS. The latent phase
can be subdivided into two stages: a condemned stage, during which
the cell is proceeding on a pathway toward death but can still be
rescued if it is exposed to antiapoptotic activities, and a committed
stage, beyond which rescue is usually impossible.

process of autodigestion and dissolution, until it spills its

cytoplasmic contents out into the surroundings (Fig.
46.3). This produces local inflammation as phagocytic
cells are activated, flock to the site, and ingest the debris
(see Figs. 22.3 and 30.13). Because agents that damage
cells act over areas that are large in comparison to the
size of a single cell, necrosis often involves large groups
of neighboring cells.
In contrast to necrosis, apoptotic cells shrink rather
than swelling and they typically do not lyse (Fig. 46.2).
Apoptosis is a two-stage process. On receipt of the
proapoptotic signal that triggers the pathway to death,
cells enter a latent phase of apoptosis (Fig. 46.4). The
duration of the latent phase of apoptosis, during which
cells appear healthy, can be extremely variable, ranging
from a few hours to several days.
FIGURE 46.5 SCANNING ELECTRON MICROGRAPH OF
INTACT AND APOPTOTIC MOUSE SARCOMA CELLS. Intact cells
are covered with microvilli, whereas apoptotic cells have numerous
smooth blebs. These cells were stimulated to undergo apoptosis as a
result of interference with RNA metabolism. (From Wyllie AH, Kerr JFR,
Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol.
1980;68:251306.)
Ultimately, the cells enter the execution phase
of apoptosis, lasting about an hour, during which
they undergo dramatic morphologic and physiological
changes. These include:
Loss of microvilli and intercellular junctions (Fig. 46.5)
Shrinkage of the cytoplasm
800 SECTION X n Cell Cycle

Dramatic changes in cytoplasmic motility with activa-

tion of violent blebbing (Fig. 46.6)
Loss of plasma membrane lipid asymmetry (see Fig.
13.7), so the distribution of phosphatidylserine is
randomized and it appears in the outer leaflet of the
membrane
Hypercondensation of the chromatin and its collapse
against the nuclear periphery
The explosive fragmentation of the cell into
membrane-enclosed apoptotic bodies that contain
remnants of the nucleus, mitochondria, and other 0 min 114 116
organelles.
All these changes are instigated by the action of a
specific set of death-inducing proteases and are discussed
at length later.
In tissues, surrounding cells rapidly phagocytose
apoptotic bodies in response to the phosphatidylserine
and other markers on their surfaces (Fig. 46.7). Apoptosis
can thus be considered to be the disassembly of the cell
into bite-sized membrane-bound vesicles, so the cel-
lular contents are not usually released into the environ- 118 120 148
ment unless phagocytosis is delayed. Surface markers on
apoptotic bodies cause cells that ingest them to secrete FIGURE 46.6 APOPTOSIS OF A TRANSFORMED PIG KIDNEY
antiinflammatory cytokines. As a result, apoptotic death CELL FOLLOWING EXPOSURE TO ETOPOSIDE, A DRUG USED
usually does not lead to an inflammatory response and IN CANCER CHEMOTHERAPY. The dramatic cytoplasmic blebbing
results in the disassembly of the cell into membrane-enclosed vesicles.
apoptotic cells seem to vanish from tissues without (Courtesy L.M. Martins and K. Samejima, Wellcome Trust Institute for
a trace. Cell Biology, University of Edinburgh, United Kingdom.)

A. Attraction of phagocytes via D

soluble find me signals
n
Mi
gration sign

Phagocyte Apoptotic
cell
al
s

B. Recognition and phagocytosis via

displayed eat me signals and
lack of dont eat me signals

C. Production of antiinflammatory
cytokines

IL-10
TGF-

FIGURE 46.7 PHAGOCYTOSIS OF APOPTOTIC CELLS. AC, Phagocytosis that occurs when cells express eat me signals results in the
production of antiinflammatory cytokines. D, Electron micrograph of a phagocytosed apoptotic body containing a nuclear fragment. The nucleus
(n) of the epithelial cell that engulfed this apoptotic body is shown at top. In this case, apoptosis occurred during allograft rejection in a pig.
(A, Modified from Lauber K, Blumenthal SG, Waibel M, et al. Clearance of apoptotic cells: getting rid of the corpses. Mol Cell. 2004;12:277287.
B, From Wyllie AH, Kerr JFR, Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol. 1980;68:251306.)
 CHAPTER 46 n Programmed Cell Death 801

Examples of Cells That Undergo
Programmed Cell Death
The following are six common causes of programmed
cell death (Fig. 46.8).
Developmentally Defective Cells
During molecular maturation of T-lymphocyte antigen
receptors (see Figs. 27.8 and 28.9), immature T cells in
the thymus (known as thymocytes) rearrange the genes
encoding the receptor and chains. To function
properly, the T-cell receptor must recognize major his-
tocompatibility complex (MHC) glycoproteins on other
thymic cells during antigen presentation (see Fig. 27.8).
T lymphocytes whose T-cell receptors cannot interact
with the spectrum of MHC glycoproteins expressed in a
given individual are ineffective in the immune response.
These cells die by apoptosis in a process known as posi-
tive selection (Fig. 46.9).
Many of the newly created receptors bind to foreign
antigens, but others interact with self-antigens. The
latter are potentially harmful and are eliminated through

A B C D
Up to 80% of
Epithelial cells must die neurons die in
to allow fusion of palate some ganglia

Mammary epithelium Over 95% of

cells die when deprived immature
of hormones at end of T cells die
lactation in thymus

Cells of Mllerian
ducts die in males E12.5 E13.5 E14.5

Prostate cells die Cells of

interdigital E
when deprived of
hormone webbing die Dying cells
(yellow)

FIGURE 46.8 PROGRAMMED CELL DEATH DURING DEVELOPMENT. A, Examples of cells that undergo programmed cell death.
BD, Programmed cell death in the embryonic mouse paw. At day 12.5 of development, the digits are fully connected by webbing. By day 13.5,
the webbing has started to die, and by day 14.5, all the webbing cells are gone. E, Cells undergoing programmed cell death take up acridine
orange, whereas cells of the surrounding healthy tissue do not. (Micrographs courtesy William Wood and Paul Martin, Department of Anatomy
and Developmental Biology, University College of London, United Kingdom.)

THYMUS PERIPHERY, SPLEEN, AND LYMPH NODES

TCR- gene Pre-TCR TCR- gene TCR T-cell

rearrangement rearrangement stimulation

Common T-cell Pro-T4 cell Immature Mature Mature T-cell T-cell blast
precursor thymocyte thymocyte

No IL-7R signal No productive No TCR signal (no Autoreactive Lack of TCR No growth-
TCR- gene positive selection) TCR (negative signal factor signals
rearrangement No productive TCR- selection) Strong TCR
gene rearrangement restimulation

APOPTOSIS

FIGURE 46.9 EXAMPLES OF SIGNALS THAT PROMOTE DIFFERENTIATION OR PROGRAMMED CELL DEATH OF IMMATURE
THYMOCYTES IN THE THYMUS AND MATURE T CELLS IN THE PERIPHERY. Thymocytes that make functional T-cell receptors (TCRs)
and do not recognize self-antigens mature, provided that they receive survival signals, such as interleukin-7. Pro-T cells are also known as
double-negative thymocytes (referring to their lack of the two cell-surface markers CD4 and CD8). Immature thymocytes express CD4 and CD8
and are known as double-positive thymocytes. Thymocytes undergo apoptosis if they produce defective T-cell receptor, recognize self-antigens,
suffer DNA damage, or receive a death stimulus (glucocorticoid hormone). More than 98% of immature thymocytes die without leaving the thymus.
(Modified from Strasser A. The role of BH3-only proteins in the immune system. Nat Rev Immunol. 2005;5:189200.)
802 SECTION X n Cell Cycle
apoptosis in a process known as negative selection
(Fig. 46.9). The drug cyclosporine, which inhibits apop-
tosis in thymocytes, can cause autoimmune disease.
Overall, defects in T-cell receptor assembly are extremely
common, and up to 98% of immature T cells die by
apoptosis without leaving the thymus.
Similar positive and negative selection steps occur
during the maturation of B lymphocytes (see Fig. 28.10),
which is accomplished by a combination of gene rear-
rangements and facilitated mutagenesis. B lymphocytes
expressing antibodies directed against self-antigens or
producing antibodies whose affinity for antigen is below
a critical threshold are eliminated through apoptosis.
Excess Cells
Programmed cell death is also widely used for quality
control during development. For example, in the brain,
embryonic ganglia often have many more neurons than
are required to enervate their target muscles. Production
of excess cells is part of a Darwinian strategy to ensure
that a sufficient number of axons reach their targets.
Programmed cell death eliminates excess neurons that
fail to make appropriate connections. Up to 80% of
neurons in certain developing ganglia die in this way.
Because of the importance of apoptosis during its devel-
opment, the brain is often seriously affected in mice
engineered to lack components of the apoptotic pathway.
Cells That Serve No Function
The elimination of obsolete cells whose function has
been completed is most evident in organisms, such as
insects and amphibians that undergo metamorphosis
during development. For example, a burst of thyroid
hormone initiates programmed cell death for resorption
of the tadpole tail.
Mammals also use programmed cell death to eliminate
obsolete tissues during development. For example,
during human embryonic development, the digits of
hands and feet are connected by a tissue webbing that
is eliminated by programmed cell death (Fig. 46.8).
During craniofacial development, the hard palate
develops from two lateral precursors, each covered in a
protective layer of epithelial cells. As the two halves
grow together at the midline of the nasopharynx, they
remain separated by this epithelial covering until, in
response to a developmental cue, the epithelial cells at
the midline undergo programmed cell death. Then the
two halves of the palate can fuse. Failure of the epithelial
cells to die at the appropriate time can interfere with the
fusion of the bone, causing cleft palate.
Populations of cells that are fully functional may
become obsolete as a result of physiological changes
in the status of an organism. For example, in male
mammals, the prostate and other accessory glands of the
reproductive system are regulated by the levels of circu-
lating male hormone. If hormone levels fall below a
critical threshold, these organs virtually disappear in a
relatively brief time as their constituent cells undergo
massive apoptotic death. Should levels of circulating
androgens rise again, the remaining prostatic stem cells
proliferate and reconstruct the gland. A similar cycle of
growth and involution is seen in the mammary gland of
female mammals, which exhibits substantial differences
in size and cellular composition in the lactating and
nonlactating states. Interference with survival signaling
by sex hormones is one important strategy that is com-
monly used in the treatment of breast and prostate cancer.
Programmed cell death also eliminates certain popula-
tions of cells that never serve any function. The mllerian
ducts develop into the female oviduct. Male embryos
also develop progenitors of these ducts, which serve no
purpose and are eliminated by apoptosis.
Cells With Perturbed Cell Cycles
Chapters 40 to 43 describe how biochemical circuits
called checkpoints regulate the cell cycle. Cells respond
to DNA damage by blocking cell-cycle progression while
attempting to repair the DNA. The p53 transcription
factor, an important downstream checkpoint EFFECTOR
induces the expression of genes encoding proteins that
arrest the cell cycle. p53 also turns on genes encoding
proteins that induce cell death. It is generally thought
that if the damage cannot be repaired quickly, the pro-
death factors win out, and the outcome is apoptosis.
Double-strand DNA breaks induced by ionizing radiation
and DNA damage induced by chemotherapeutic agents
frequently result in cell death rather than repair.
A second important cell-cycle checkpoint regulates
the transition from the G1 phase to S phase. Passing the
restriction point (see Fig. 41.8) represents the commit-
ment of the cell to undergo another cycle of DNA repli-
cation and division. Restriction point control centers on
the regulation of the E2F family of transcription factors.
E2F regulates genes that promote cell-cycle progression.
E2F also regulates the expression of genes that promote
apoptosis. If activated too strongly, E2F can initiate
apoptosis as, for example, after DNA damage (see Fig.
41.14) or when restriction point control has broken
down. Cells that die in response to inappropriate
signals to proliferate include those infected by certain
viruses or overexpressing genes that drive cell prolifera-
tion (such as c-myc and c-fos [see Fig. 46.15]). Apoptotic
death of cells with an inappropriate stimulus to prolifer-
ate is an important defense against cancer.
Virus-Infected Cells
Cytotoxic T lymphocytes eliminate virus-infected cells
by causing them to undergo programmed cell death
either by apoptosis or by a second related pathway. For
example, programmed cell death accounts for at least
part of the loss of mature CD4+ T-helper cells (see Fig.
28.9) in people infected with HIV-1. When exposed to

agents that normally stimulate cell proliferation, these
cells instead undergo apoptosis. Paradoxically, it appears
that many of the dying cells are not themselves infected
with HIV.
Chemotherapeutic Killing of Cells
Exposure of cancer cells to many of the agents used in
chemotherapy does not kill the cells outright. Instead,
they die because the drugs cause intracellular damage
that acts as a signal for the induction of apoptotic cell
death. Thus, the treated cancer cells kill themselves.
Genetic Analysis of Apoptosis
Several key components that are involved in the apop-
totic execution of mammalian cells were discovered by
genetic analysis of the nematode worm Caenorhabditis
elegans. Because C. elegans is optically clear, it is pos-
sible to see every cell in a developing worm using dif-
ferential interference contrast optics (see Fig. 6.2). This
enabled investigators to trace the lineage of every cell in
an adult worm back to the fertilized egg. These studies
led to the surprising discovery that programmed cell
death is one of the most common fates for newborn C.
elegans cells. Of the 1090 somatic cells that are produced
during embryogenesis of the C. elegans hermaphrodite,
131 undergo programmed cell death at reproducible
locations and times.
Mutations in at least 14 C. elegans genes affect pro-
grammed cell death (Fig. 46.10). These are divided into
three classes: (a) genes that mark cells for subsequent
programmed death; (b) genes that are involved in cell
killing and its regulation; and (c) genes that are involved
egl-1 Determination
ced-9 ced-3
ced-4 Killing
Dead cell
ced-1, -6, -7
ced-2, -5, -10, -12 Engulfment
nuc-1 Degradation
Mammalian homologs
ced-9 protein = Bcl-2 family (antiapoptotic)
egl-1 protein = Bcl-2 family (BH3-only proapoptotic)
ced-4 protein = Apaf-1
ced-3 protein = initiator caspases
FIGURE 46.10 GENETIC DISSECTION OF PROGRAMMED
CELL DEATH. The ced (cell death abnormal) mutants of the nematode
worm Caenorhabditis elegans affect the killing, engulfment, and deg-
radation stages of programmed cell death.
CHAPTER 46 n Programmed Cell Death 803
in the phagocytosis and subsequent processing of the
cell corpses. These mutants are collectively known as
cell death abnormal (ced) mutants. Interestingly, this
repertoire of nematode genes is vastly simplified from
the apoptosis genes in the last eumetazoan common
ancestor (see Fig. 2.8). By contrast, humans have
approximately 300 genes involved in apoptosis.
The three best-known worm cell death genes are ced-
3, ced-4, and ced-9. If either ced-3 or ced-4 is inactivated,
all cells throughout the organism that should die by
apoptosis are reprieved. These cells remain alive and are
apparently functional. Interestingly, these worms have
normal life spans. This suggests that programmed cell
death is not involved in the normal aging process, at least
not in C. elegans. Ced-9 negatively regulates ced-3 and
ced-4. In worms with ced-9 loss-of-function mutations
many cells die that normally stay alive. This is deleterious
for the organism, so ced-9 mutants die. The key gene
triggering apoptosis is egl-1. Its regulation appears to
govern apoptosis in the worm. All these genes have
mammalian counterparts (discussed more fully later).
Ced-3 is a member of a specialized family of cell
death proteases called caspases. Ced-4 is a scaffolding/
adapter protein that plays an essential role in the activa-
tion of Ced-3 from its zymogen precursor. The mamma-
lian counterpart of Ced-4 is apoptotic protease-activating
factor-1 (Apaf-1). Ced-9 is a member of the Bcl-2 family
of cell death regulators (Fig. 46.14). Some Bcl-2 family
members protect against cell death, but others actively
promote cell death. Egl-1 is a BH3-only proapoptotic
regulator.
Phagocytosis turned out to be surprisingly complex,
with eight C. elegans genes encoding proteins that
are involved in the engulfment and processing of cell
corpses. Several are signaling proteins that reorganize
the cytoskeleton to permit the cell to move toward and
engulf its target. Another, the nuc-1 gene, encodes one of
several nucleases that digest the DNA of the dead cell in
lysosomes of cells that ingest the corpse. In mammals, this
digestion typically is initiated within the dying cell itself.
Signals and Pathways of Apoptosis
Two principal pathways lead to cell death by apoptosis.
These are introduced only briefly here and expanded
upon later. The intrinsic pathway (see Fig. 46.17) is
activated by internal surveillance mechanisms or signals
sent (or not sent) by other cells. Signals that induce this
pathway include DNA damage, exposure to chemicals
that interfere with a variety of cellular pathways and
excessive activation of factors that promote cell-cycle
progression. Withdrawal of nutrients or survival signals
from the environment also activates the intrinsic
pathway. Those survival signals include lymphokines,
such as interleukin-2 and interleukin-3, which are essen-
tial for survival of thymocytes; nerve growth factor,
which is required for survival of many neurons; and
804 SECTION X n Cell Cycle
extracellular matrix, which is required for survival of
epithelial cells. Signals that activate the intrinsic pathway
converge on mitochondria, which release key factors
that drive the apoptotic response.
Signals from other cells are the primary triggers of the
extrinsic pathway (see Fig. 46.18). Direct contact of a
killer cell with the target cell activates specific receptors
that initiate this pathway, starting at the plasma mem-
brane. Activation of the extrinsic pathway is one strategy
that cytotoxic T lymphocytes use to kill cells that are
recognized as foreign (or as harboring foreign patho-
gens). This pathway is also widely used to control cell
populations in the immune system. In many cells, the
extrinsic pathway activates the intrinsic pathway, which
is then responsible for killing the cell.
Protein Regulators and Effectors of Apoptosis
Because the penalty for misregulation of apoptosis is
death, it is not surprising that the process is carefully
regulated. This is essential for cells but complicates
matters for students. This section first lays out the
overall strategy in generic terms and then fills in some
important details.
A cascade of proteases called caspases drives apop-
tosis. Each caspase is harmless until activated (usually by
dimerization for initiator caspases and proteolytic cleav-
age for effector caspases). Activation of a small number
of initiator caspases triggers a cascade that amplifies the
response and results in activation of numerous effector
caspases. The ability of effector caspases to activate
further initiators and effectors further amplifies the
response.
This strategy of employing amplification and positive
feedback has two powerful advantages. First, it can
provide a very rapid change in the state of the cytoplasm,
from pro-life to pro-death within seconds. Second, the
relatively small number of initiator caspases that trigger
the cascade constitutes a feasible target for negative regu-
lators that can rapidly quell responses that are initiated
under borderline conditions or by mistake. This is benefi-
cial but also complicates the overall system. If initiator
caspases start apoptosis and are then inactivated by
suppressers, how does the response ever take hold? The
answer is at least one more level of regulation: inhibitors
of the inhibitors. This additional layer of regulation is
thought to enable the rapid burst of caspase activation
that triggers the onset of apoptotic execution.
The following sections discuss the workhorses of
apoptosisthe caspasesfollowed by regulation of the
response.
Caspases
Caspases (cysteine aspartases) are specialized proteases
with a cysteine in their active site that cleave their targets
on the C-terminal side of aspartate residues. Caspases
generally inactivate cellular survival pathways and
specifically activate other factors that promote cell death.
In certain specialized instances caspases can participate
in pathways leading to activation of nuclear factor B
(NF-B; see Figs. 10.21 and 27.8) and the immune
response and also in terminal differentiation. These
nonlethal roles of caspases are not discussed further here.
C. elegans has three caspases, one of which (Ced-3)
is essential for cell death. In contrast, mammals have as
many as 17 caspase-related genes (Fig. 46.11A). Analysis
based on sequence comparisons divides caspases into
two major subfamilies. The caspase 1 subfamily encodes
enzymes that process prointerleukin-1 to yield mature
interleukin-1 and prointerleukin-18 to yield mature
interleukin-18. Macrophages secrete these cytokines,
which cause fever and inflammation. The second caspase
3 subfamily of enzymes participates almost exclusively
in apoptotic cell death.
Like many proteases, caspases are synthesized as
inactive zymogens, known as procaspases. All living
vertebrate cells synthesize procaspases constitutively.
Procaspases typically consist of three domains: an N-
terminal prodomain followed by the large and small
subunits of the mature enzyme (Fig. 46.11AB). Aspar-
tate residues between these three domains are targets
for cleavage by caspases. Procaspases are activated either
by multimerization or by cleavage and release of the
prodomains. Following procaspase cleavage, the two
large and two small subunits associate in a compact,
block-like heterotetrameric molecule (Fig. 46.11BD).
Depending on the enzyme, multimerization or cleavage
of the procaspase permits a major conformational change
in the polypeptide, creating two stable active site pockets
between the large and small subunits.
Two classes of caspases are involved in cell death.
Initiator caspases have long prodomains (Fig. 46.11A).
They exist as monomers in cells and become autoactivated
when scaffolding cofactors promote their aggregation.
Activation is induced by dimerization and does not require
proteolytic cleavage (although cleavage typically follows
activation). Sequences within the extended prodomains
are involved in targeting the initiator procaspases to the
appropriate cellular locations and in interactions with
scaffolding factors.
Effector caspase zymogens are monomers with
short prodomains. These inactive enzymes are typically
incapable of autoactivation. Instead, they are activated
through cleavage by initiator caspases or by other active
effector caspases.
Scaffolding proteins and adapters play an essential
role in activating initiator caspases by forming multi-
protein activation platforms. For the intrinsic pathway
of cell death, factors released from mitochondria (dis-
cussed later) activate the scaffold protein Apaf-1, which
is related to AAA ATPases (adenosine triphosphatases)
(see Box 36.1). Active Apaf-1 forms a ring-like structure
with seven spokes called the apoptosome (Fig. 46.17).
 CHAPTER 46 n Programmed Cell Death 805

~p20 ~p10
A B. Caspase maturation
Effector caspases 0 28 175 277
Casp-3 LS SS Procaspases (zymogens) Active enzyme
303 Pro
Casp-7 LS SS Prodomain
DED DED LS SS
293
Casp-6 LS SS Caspase 8 (an initiator caspase)

Initiator caspases 479

Casp-8 DED DED LS SS LS SS

521 Caspase 3 (an effector caspase)

Casp-10 DED DED LS SS
416
Casp-9 Card LS SS
435 C. Caspase 1 D. Caspase 3
Casp-2 Card LS SS

Proinflammatory caspases 377

Casp-4 Card LS SS
418
Casp-5 Card LS SS
373
Casp-11 Card
419
Casp-12 Card
404
Casp-1 (ICE) Card LS SS
242
Casp-14

FIGURE 46.11 INTRODUCTION TO CASPASES. A, The 13 enzymatically active mammalian caspases fall into three groups. Where it has
been determined, the portions of the zymogens that give rise to the large (blue) and small (yellow) subunits are shown. B, Initiator caspases have
large prodomains that participate in subcellular targeting. Two initiator procaspases come together to form the active enzyme. CD, Ribbon
diagrams of crystal structures of caspases 1 and 3. The catalytic residues come primarily from the large subunit (blue). The space-filling structure
(red) represents a peptide inhibitor covalently bound in the active site of the enzyme.

Binding of procaspase 9 zymogen to this platform pro-
motes dimerization and activation of the enzyme.
The scaffold proteins for the extrinsic death pathway
are cytoplasmic domains of cell-surface receptors. When
these receptors bind their ligands on the surface of other
cells, they form stable trimeric complexes that recruit
adapter proteins to form a signaling complex called the
death-inducing signaling complex (DISC). Interac-
tions involving multiple proteinprotein interaction
motifs link the procaspase 8 zymogen to the DISC (Box
46.2), resulting in dimerization, activation, self-cleavage,
and release of active caspase 8.
Caspases cleave as many as 1000 cellular proteins
(Fig. 46.12). Some targets are structural proteins, but
many are involved in cellular signaling. For example,
caspases cleave several protein kinases. Many kinases
have pseudosubstrate motifs that autoinhibit the kinase
and can be moved out of the way in response to physi-
ological stimuli (see Fig. 25.4). Caspase cleavage often
neatly clips off these regulatory domains, thereby pro-
ducing constitutively active enzymes. These unregulated
kinases may alter signaling pathways to promote cell
death. Caspases also cleave and inactivate several pro-
teins that normally function in the detection and repair
of DNA damage.
Caspases can also create factors that directly promote
cell death. The most obvious example is caspase activa-
tion of other caspases in the death cascade. Caspases
also act indirectly to cause mitochondria to release
factors that promote cell death through the intrinsic
pathway. Caspase cleavage of an inhibitory chaperone is
responsible for activation of the nuclease that ultimately
destroys the chromosomal DNA of most cells undergoing
apoptosis (see section on CAD nuclease).
Natural Caspase Inhibitors
Because most healthy cells express initiator procaspases
with the potential to oligomerize and kill the cell, it is
important to have a mechanism that dampens any poten-
tial noise in the pro-apoptotic pathway. The inhibitor
of apoptosis protein (IAP) family is defined by the pres-
ence of a motif of approximately 80 amino acids known
as a baculovirus IAP repeat (BIR) domain. This is a
type of Zn2+ finger (see Fig. 10.14) that mediates protein
protein interactions. IAP proteins inhibit caspases in two
ways. First, they bind the caspase and invade the active
site, thereby blocking its access to substrates. Second,
several IAPs are E3 ubiquitin ligases (see Fig. 23.3) that
ubiquitylate caspases and tag them for destruction by
proteasomes.
If IAP proteins inactivate caspases, then how is the
apoptotic response ever initiated? Cells also express
several molecules that can bind and inactivate IAPs. The
best characterized of these, is the second mitochondrial
activator of caspases (Smac or DIABLO). Smac is nor-
mally sequestered in mitochondria, but is released into
806 SECTION X n Cell Cycle

BOX 46.2 Matchmaking for Cell Death: the Key Is in the Domains

There are so many different proteins involved in apoptosis
that even experts have a difficult time keeping them all
straight. However, an understanding of the general principles
is much simplified if we recognize that most proteins
involved in apoptosis regulation are built from a relatively
limited number of modules, which act as sites for protein
protein interactions. The following are the most important
modules for apoptosis.
Bcl-2 family members are defined by the presence of
short regions of conserved sequence, referred to as BH
domains (Bcl-2 homology). One of these, the approxi-
mately 20-residue BH3 domain, found in all Bcl-2 family
members, promotes complex formation between Bcl-2
family members by docking into a so-called BH3 binding
groove.
Four domains regulate caspase targeting and activation.
Although the amino acid sequences of these domains have
diverged extensively, all are regions of approximately 80 to
90 residues that form a characteristic bundle of six -helices.
These domains all were present in the last eumetazoan
common ancestor.
1. The death domain (DD) is found in many proteins that
are involved in signaling pathways related to cell death.
These include cell death receptors, such as Fas (and
others), and adapter molecules, such as FADD (Fas-
associated death domain).
2. The death effector domain (DED) is found in adapters
such as FADD, the prodomains of caspases 8 and 10, and
certain inhibitors of apoptosis.
3. The Pyrin domain (PYD) is found in proteins involved
in inflammation (such as microbial sensors) and the
activation of caspase 1.
4. The caspase recruitment domain (CARD) is found in
several adapter proteins involved in cell death, including
CED-4 and Apaf-1, and in caspases 1, 2, and 9.
The domains have similar folds, but different distributions
of surface charge. As a result, they prefer to interact with
themselves (ie, DDDD, DEDDED, PYDPYD, and CARD
CARD). Such interactions are said to be homophilic. When
a new apoptosis effector protein is identified, it is possible
to predict from an analysis of its amino acid sequence which
of the known proteins it is most likely to interact with.

A. Inactivation of protective factors and activation B. Pathway of nuclear apoptosis CYTOPLASM

of proapoptotic factors by caspases
Initiator Effector Cytoplasmic
caspases caspases targets
(8, 9, etc.) (3, 6, 7, etc.)
p53 Focal
pRb Surface Ras
mdm2 receptors contact RasGAP
Raf FAK Paxillin
Actin
Cdk/cyclins Gas2
active MEK PP2A Cb1/1b CAD / Caspase-6
p27 ICAD PARP
p21 Wee1 Lamins
APC PI3K
A A
Cdk/cyclin ERK ICAD/ I CA D CAD P RP L M I NS
inactive NF-B DFF45 Akt Domain NUCLEUS
STAT1 nuclease
CAD Death Bad
Cell-cycle ? Bcl-2 Fragmentation
arrest ? nuclease
PKC Bcl-XL
Survival PKC JNK-p38 Bid

MEKK1 PAK2
Mst1/Krs

FIGURE 46.12 SOME WAYS THAT CASPASES PROMOTE CELL DEATH. A, A few of the many proteins cleaved by caspases in apoptotic
cell death. Proteins shown in green normally have a role in keeping the cell alive and are inactivated by caspases. Proteins shown in red are
turned into active death-promoting factors as a result of caspase cleavage. Proteins shown in black are not cleaved and are included to show
the pathways that are affected by cleavage. Caspases inactivate a number of pathways that promote cell survival, thereby strongly reinforcing
the decision of the cell to die. B, Some of the roles of caspases in disassembly of the nucleus. CAD, caspase-activated deoxyribonuclease; ERK,
extracellular signal-regulated kinase; ICAD, inhibitor of caspase-activated deoxyribonuclease; JNK, c-Jun N-terminal kinase; MEK, mitogen
activated protein/extracellular signal-related kinase kinase; NF-B, nuclear factor B; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase
C; STAT, signal transducer and activator of transcription.

the cytoplasm when the intrinsic pathway of apoptosis
is initiated as a result of permeabilization of the mito-
chondrial outer membrane. It then binds to the BIR
domain, inactivating the IAPs. Mathematical modeling
shows that inactivation of IAPs by Smac is needed to
make the proapoptotic signal act like a switch rather
than a graded response.
IAPs were discovered in studies of the mechanisms
viruses use to avoid being eliminated by cell death.
When viruses infect cells and disassemble their capsids,
they become vulnerable to suicide defense mechanisms:
If cells can kill themselves before the virus has time to
complete its life cycle, they will take the virus with them,
and the organism will survive. To defend against this,
 CHAPTER 46 n Programmed Cell Death 807

viruses pilfer genes encoding cellular proteins and adapt
them for their own means. For example, insect baculo-
viruses make two proteins that inhibit apoptosis, keeping
the cell alive long enough for the virus to reproduce.
One of these, IAP, was derived from a cellular gene. The
origin of the second IAP inhibitor, p35, is less clear. p35
is a broad-spectrum caspase inhibitor that is thought to
work by a serpin-like mechanism. Serpins are special
protease substrates that, after cleavage, form a tight
complex with the enzyme and inactivate it. Several
mammalian pox viruses also make a serpin-like inhibitor
of certain caspases called CrmA.
CAD Nuclease and Its Chaperone ICAD
The chromosomal DNA is destroyed during apoptosis.
The nucleases involved in cleaving the cellular DNA
during (and after) apoptotic cell death fall into two
classes. Cell autonomous nucleases degrade the DNA
within the dying cell (Fig. 46.13A). The best known is

A. DNA cleavage during apoptosis B. Active CAD nuclease

DNA

Proapoptotic Execution Engulfment Clearance

stimulus
Large Nucleosomal Further digestion
fragments fragments within phagocytic cell

Cell autonomous Waste management

nucleases nucleases

Necrosis Lysis Extracellular digestion

C. ICAD regulates CAD function

D. Drug-induced DNA E. Effect of purified CAD on
Apoptotic stimuli cleavage in vivo isolated nuclei in vitro
Caspase activation
23
CAD / Inactive 9.4
ICAD ICAD 6.6

Active
CAD 2.0
ICAD Dimerized CAD
protein cleaves DNA

0.56
CAD mRNA
CYTOPLASM NUCLEUS 3 ICAD/CAD
0 1 2 3 4 6h 1 2 3 4 + Casp-3

FIGURE 46.13 NUCLEASES DIGEST THE CELLULAR DNA DURING APOPTOSIS. A, In apoptosis, the DNA is digested first to large
fragments and later to nucleosome-sized pieces (see Fig. 13.1) by cell autonomous nucleases expressed within the dying cell. Waste management
nucleases made by other cells also have an essential role in cleaning up apoptotic and necrotic debris. B, The predominant cell autonomous
nuclease (CAD) has a scissors-like structure. C, ICAD is an inhibitory chaperone for CAD, promoting its folding on the ribosome and continuing
as an inhibitor when CAD is stored in the nucleus. ICAD cleavage leads to CAD activation. D, Cleavage of the chromosomal DNA by CAD during
chemotherapy-induced apoptosis of a leukemia cell line. DNA separated according to size by electrophoresis on an agarose gel was stained with
ethidium bromide. The ladder of bands reflects cleavage between adjacent nucleosomes. E, Activated CAD causes chromatin condensation and
appearance of an apoptotic morphology in isolated cell nuclei. Cloned CAD and ICAD were expressed together in Escherichia coli and incubated
with nuclei. ICAD cleavage with caspase 3 released active CAD, which degrades the nuclear DNA (Lane 3). Other lanes: DNA gel size markers
(left); nuclei incubated with buffer or caspase 3 alone (Lanes 1 and 2, respectively); same experiment as in Lane 3 but performed by using a
mutant ICAD that could not be cleaved by caspase 3 (Lane 4). To the right is an electron micrograph of a thin section of one nucleus with
condensed chromatin at the nuclear periphery. CAD, caspase-activated deoxyribonuclease; ICAD, inhibitor of caspase-activated deoxyribonucle-
ase; mRNA, messenger RNA. (A, Modified from Samejima K, Earnshaw WC. Trashing the genome: The role of nucleases during apoptosis. Nat
Rev Mol Cell Biol. 2005;6:677688. B, For more information, see Protein Data Bank [PDB; www.rcsb.org] file 1V0D from Woo EJ, Kim YG, Kim
MS, et al. Structural mechanism for inactivation and activation of CAD/DFF40 in the apoptotic pathway. Mol Cell. 2004;14:531539. D, From
Kaufmann SH. Induction of endonucleolytic DNA cleavage in human acute myelogenous leukemia cells by etoposide, camptothecin, and other
cytotoxic anticancer drugs: a cautionary note. Cancer Res. 1989;49:58705878. E, Courtesy K. Samejima, Wellcome Trust Institute for Cell
Biology, University of Edinburgh, United Kingdom.)
808 SECTION X n Cell Cycle

the caspase-activated DNase (CAD; see later discussion). Bcl-2 family

A mitochondrial nuclease known as endonuclease G may
also degrade DNA in some cell types. Cell autonomous Protectors Killers Regulators
nucleases are dispensable for cell death and for the life Bcl-2 Bax Bad Bik / Nbk
Bcl-xL Bak Bid HRK
of the organism. They might have evolved to eliminate Bcl-W Bok / Mtd Bim Puma
viral DNA as part of the suicide defense response Mcl-1 Bcl-xS BmF Noxa
described in the previous section. A1 Hrk
Boo/Diva C. elegans Egl-1
Waste management nucleases clean up the debris C. elegans ced-9
after cells die. They either function within lysosomes of Adenovirus E1B19K
cells that have ingested apoptotic cell fragments or are Epstein-Barr virus BHRF1
secreted into the extracellular space. DNase II, one of
the most important waste management nucleases, is
essential for life. Mouse embryos that lack DNase II BH4 BH3 BH1 BH2 Protectors
become overwhelmed with undegraded DNA and die.
Cell autonomous nucleases act in two stages. After BH3 BH1 BH2 Killers
an initial cleavage of the chromosomes into fragments
of roughly 50,000 base pairs, DNA is usually cleaved BH3 Regulators
between nucleosomes, producing a characteristic
ladder of DNA fragments with a periodicity of approxi-
mately 200 base pairs. This ladder is seen when DNA FIGURE 46.14 THE BCL-2 FAMILY OF APOPTOTIC REGULA-
TORS. C. elegans, Caenorhabditis elegans.
isolated from apoptotic cells is subjected to gel electro-
phoresis. The responsible nuclease is CAD. CAD is nor-
mally present in a complex with ICAD (inhibitor of CAD normally survive into the adulthood. A Ced-9 mutation
[Fig. 46.13C]). The complex of CAD and ICAD is also kills the worm. Human Bcl-2 is functionally and structur-
known as DNA fragmentation factor (DFF). ICAD is a ally homologous to C. elegans Ced-9 and can substitute
chaperone required to fold CAD into an active conforma- for it in living worms. This ability of a human gene to
tion during translation on the ribosome. However, ICAD protect nematodes reveals that the fundamental machin-
also inhibits the nuclease activity of CAD. This dual func- ery of apoptotic cell death has been conserved over great
tion of ICAD guarantees that only inactive CAD can be evolutionary distances.
synthesized in healthy cells. During apoptosis, caspase 3 Bcl-2 family members are defined by the presence of
cleaves ICAD and releases active CAD nuclease. one to four short blocks of conserved protein sequence
called BH (Bcl-2 homology) domains. Antiapoptotic
Bcl-2 Proteins and the Intrinsic Pathway Bcl-2 protectors typically have four of the domains.
Proapoptotic Bcl-2 killers typically have three of these
of Apoptotic Cell Death
domains, while the Bcl-2 regulators have only the BH3
As mentioned previously, mitochondria are key players in domain. The BH3 domain is a short helix that fits into
a pathway to cell death that is triggered by a variety of a groove on the surface of both Bcl-2 protectors and
toxic insults (Fig. 46.17). The Bcl-2 family of proteins killers, forming complexes that regulate their activity. It is
regulates these mitochondrial events. The following sec- believed that the Bcl-2 protectors regulate the behavior of
tions describe this important protein family and the regu- Bcl-2 killers by a similar interaction. For example, the Bcl-2
lation of the intrinsic pathway of apoptosis. protein forms a complex with a proapoptotic Bcl-2 killer
called Bax, thereby interfering with the ability of Bax to
Bcl-2 Proteins kill cells. Binding of BH3-only proteins to Bcl-2 protectors
Bcl-2 proteins can be grouped into three subfamilies (Fig. can inactivate their antiapoptotic functions. Egl-1 is a
46.14). Bcl-2 protectors protect cells against apoptosis. BH3-only protein and this is how it triggers apoptosis. A
Bcl-2 killers (eg, Bax and Bak) are proapoptotic proteins new generation of BH3 mimetic drugs induces apoptosis
that actively kill cells. Bcl-2 regulators (widely known as of cancer cells by mimicking this second mechanism.
BH3-only proteins) promote cell killing by either interfer- Genetic experiments in mice revealed several differ-
ing with the protectors or activating the killers. Bcl-2 ent functions for Bcl-2 family members. Mice born
proteins primarily regulate the release of death-promoting without Bcl-2 have deficiencies of the immune system
factors from mitochondria when cells receive signals that are best understood if one role of this protein in vivo
that activate the intrinsic pathway. is to render lymphocytes resistant to proapoptotic signals
C. elegans genetics identified a gene, ced-9, that pro- during immune system maturation. Mice lacking another
tects cells against apoptosis and another gene, egl-1, pro-life family member, Bcl-xL, die during embryogenesis,
that inactivates ced-9 protein and triggers apoptosis. In apparently as a result of widespread death of neurons
ced-9 mutants, many cells die during development that in the central and peripheral nervous systems and

hematopoietic cells in the liver. In contrast, loss of the
killers Bax plus Bak makes cells highly resistant to apop-
tosis by a wide variety of intrinsic pathway stimuli.
Bcl-2 Family Members and Cancer
A gene that prevents cells from dying poses a potential
danger in multicellular organisms, in which rates of cell
proliferation and death must be balanced carefully. In
fact, the name Bcl-2 comes from the discovery that this
gene is the culprit responsible for certain types of B-cell
lymphoma. These particular lymphomas arise when a
chromosome translocation, involving chromosomes 14
and 18, moves the Bcl-2 gene into the immunoglobulin
heavy-chain gene cluster, a site of very active gene
transcription in B lymphocytes. Elevated transcription of
Bcl-2 is thought to be directly responsible for the cancer-
ous phenotype in these patients, making Bcl-2 a cancer-
promoting oncogene (see Chapter 41).
Unlike many other oncogenes, Bcl-2 overexpression
does not cause cell proliferation. Instead, it disrupts
the balance of regulation between life and death. Cells
that overexpress Bcl-2 protein actually grow somewhat
slower than normal but are highly resistant to many
stimuli that normally promote cell death. The net result
is an accumulation of B cells: a lymphoma. Other anti-
apoptotic Bcl-2 family members are amplified in a variety
of solid tumors and loss of microRNAs directed at Bcl-1
is thought to play a major role in development of chronic
lymphocytic leukemia. Fig. 46.15 shows an example of
Bcl-2 conferring resistance to death when the cell cycle
is perturbed by expression of an oncogene.
Intrinsic Pathway of Apoptotic Death
In addition to their role in energy production, mitochon-
dria have an essential role as sensors of the health of the
cell. If cells sense insults from which they cannot recover,
mitochondria trigger the intrinsic pathway of cell
death (Fig. 46.17). Bcl-2 family members regulate this
pathway. The regulation seems straightforward in C.
elegans, in which the protector CED-9 (Bcl-2like) binds
to the CED-4 scaffolding protein (Apaf-1-like) and inter-
feres with its activation of the CED-3 caspase. Apoptosis
is induced when the regulator BH3-only protein EGL-1
binds to CED-9 and blocks it from inactivating CED-4.
In mammals, the situation is more complex, partly
because Bcl-2 family members are more numerous and
partly because they do not interact in such a straightfor-
ward fashion. In mammals, two of the killer proteins,
Bax and Bak, are essential for activation of the intrinsic
pathway. In healthy cells, Bak is loosely associated
with the mitochondrial outer membrane, and Bax is in
the cytoplasm (Fig. 46.17). On receipt of a proapoptotic
stimulus, Bax and Bak insert deeply into the mitochon-
drial outer membrane and form oligomeric membrane
CHAPTER 46 n Programmed Cell Death 809
100
Cell viability (% control)
80
60 Normal levels of c-myc
40
Overexpressed c-myc
and overexpressed Bcl-2
20
Overexpressed c-myc
0
0 30 60 90 120
c-myc induction (min)
FIGURE 46.15 BCL-2 PROTECTS CELLS FROM ONCOGENE-
INDUCED CELL DEATH. Chinese hamster ovary cells die when they
are induced to express abnormally high levels of the c-myc protein,
but simultaneous expression of the Bcl-2 protein rescues them from
this effect. These cells contain many copies of the c-myc gene under
control of a promoter that is activated when the cells are briefly
exposed to high temperature (43C for 90 minutes). The curves show
the percentage of viable cells remaining at various times following the
induction of c-myc expression. The Bcl-2 gene was introduced into
these cells on a plasmid under the control of a promoter that is always
active. The blue line represents the parental cell line lacking either the
cloned c-myc or Bcl-2 genes. (Note that approximately 40% of these
cells die following the heat treatment used to induce c-myc expres-
sion.) The yellow line shows that the cells that produce high levels of
c-myc protein alone rapidly die (by apoptosis). The red line shows that
cells expressing both the c-myc and Bcl-2 proteins survive the treat-
ment almost as well as the parental cells. (From Bissonnette RP,
Echeverri F, Mahboubi A, et al. Apoptotic cell death induced by c-myc
is inhibited by bcl-2. Nature. 1992;359:552554.)
pores that allow the release of pro-apoptotic factors from
the mitochondrial intermembrane space. Binding of
antiapoptotic Bcl-2 family members to Bax/Bak somehow
prevents mitochondrial outer membrane permeabiliza-
tion. BH3-only family members can either directly
promote death by facilitating Bax/Bak oligomerization or
indirectly promote it by binding and neutralizing anti-
apoptotic Bcl-2 family members.
The proapoptotic factors released from the mitochon-
drial intermembrane space by Bax and Bak include the
electron transport protein cytochrome c (see Fig. 19.5),
Smac, and endonuclease G (Fig. 46.16). These mitochon-
drial proteins actively promote apoptotic cell death. In
the cytoplasm, cytochrome c binds to the scaffolding
protein Apaf-1, a mammalian homolog of C. elegans
CED-4 protein, causing it to undergo a conformational
change that exposes a hidden bound adenosine diphos-
phate (ADP) to solvent. Exchange of this ADP for deoxy-
adenosine triphosphate (dATP) allows Apaf-1 to form a
wheel-like structure with seven spokes called the apopto-
some (Fig. 46.17). Apaf-1 in the apoptosome binds caspase
9 through an N-terminal caspase recruitment domain.
Binding to the apoptosome elevates the catalytic
activity of procaspase 9 approximately 2000-fold without
the need for its cleavage. Thus, the active form of caspase
9 is an oligomeric complex of the procaspase with
the apoptosome. Activated caspase 9 then cleaves
810 SECTION X n Cell Cycle

multiple procaspase 3 zymogens, amplifying the cell

A. Life death cascade. It also cleaves itself, triggering its release
Cytochrome c
Smac, AIF from the apoptosome with a resulting loss of activity.
Intermembranous
space
Endonuclease G This autocleavage acts like a timer limiting apopto-
Others?
some activity.
Mitochondrial
outer membrane This cascade can be further amplified in at least two
permeabilization ways. First, caspase 3 cleaves other effector caspases,
directly amplifying the cascade. In addition, active cas-
B. Death pases cleave the BH3-only protein Bid, which then acti-
Bid vates more Bax and Bak in a feedback loop, thereby
Caspases
Bax promoting the release of more cytochrome c and Smac,
Other Bak
Cytochrome c
and enhancing caspase 9 activation.
Activation of signals
downstream Smac It was extremely surprising to find that an essential
caspases AIF metabolic protein such as cytochrome c has a second
Caspase 9 Endonuclease G function that is essential for death. Among the studies
activation
Other Others
supporting the Jekyll-and-Hydelike nature of this protein
targets in life and death was the engineering of mice whose
To nucleus
Nuclear Other cytochrome c can function in electron transport but
disassembly targets cannot bind Apaf-1. These mice die as a result of brain
abnormalities caused by insufficient cell death.
FIGURE 46.16 MITOCHONDRIA INTEGRATE A CELLS LIFE
AND DEATH DECISIONS. A, In healthy cells, a number of factors
that promote apoptosis are stored in the intermembrane space of the
mitochondria. B, In cells undergoing apoptosis, BH3-only proteins
Extrinsic Pathway of Apoptotic Death
trigger the proapoptotic Bcl-2 family members to induce the release Cells express at least six different cell surface molecules,
of these death-promoting factors; this initiates an amplifying cycle that
collectively termed death receptors, that can trigger
ultimately leads to cell death. AIF, apoptosis-inducing factor; Smac,
second mitochondrial activator of caspases. apoptotic death. Protein ligands on the surface of other

A. Life Metabolites up C. Apoptosome

to 10,000 D 3 (Apaf-1 scaffold)
9

Bak Activation of
Bid downstream
3 caspases
Autoactivation
VDAC
of caspase 9 (stays
3
6 bound to apoptosome)
MOMP
Caspases
cleave Bid
Bax B. Death
tBid 9
Proteins not to scale

Procaspase 9 9

Procaspase CARD domain

Apaf-1
CARD
domain Cytochrome c
dADP
Aggregation
Apaf-1 of Apaf-1 scaffold
* Conformational (apoptosome)
* *AIF
Smac, (ATP)
change in Apaf-1
endonuclease G (exchanges dADP for ATP) 27nm

FIGURE 46.17 INTRINSIC CELL DEATH PATHWAY. AB, Following activation by the BH3-only protein Bid, Bax and Bak form a pore that
releases cytochrome c. Released cytochrome c binds to Apaf-1, inducing a conformational change leading to formation of the apoptosome,
which binds and activates procaspase 9 via scaffold-induced oligomerization. Activated caspase 9 subsequently activates downstream effector
caspases, leading to cell death. C, Molecular organization of the apoptosome. (C, Modified from PDB file 3JBT from Zhou M, Li Y, Hu Q, et al.
Atomic structure of the apoptosome: mechanism of cytochrome c- and dATP-mediated activation of Apaf-1. Genes Dev. 2015;29:23492361.)
 CHAPTER 46 n Programmed Cell Death 811

cells bind and activate these receptors, turning on path-
ways that can lead to apoptotic death.
One well-characterized death receptor called Fas (also
known as Apo1 or CD95) is a member of the tumor
necrosis factor receptor family (see Fig. 24.9). Fas is a
type I membrane protein whose extracellular domain
consists of three cysteine-rich domains (Fig. 24.9 shows
the atomic structure of the related trimeric tumor necro-
sis factor receptor with bound ligand). The cytoplasmic
domain of Fas contains a death domain of approxi-
mately 80 residues, which is shared by all the death
receptors (see Box 46.2).
The Fas ligand is a trimeric 40-kD intrinsic membrane
protein found on the surface of cells. Cytotoxic T lym-
phocytes use Fas ligand to rid the body of virally infected
cells. When a cytotoxic T lymphocyte contacts a target
cell, the Fas ligand on the lymphocyte surface binds to
Fas on the target cell and initiates the extrinsic pathway
of apoptotic death (Fig. 46.18). Ligand binding activates
signaling from the intracellular death domain of Fas,
possibly by stabilizing Fas trimers or by altering their
conformation. Activated Fas binds an adapter protein
called FADD (Fas-associated protein with a death
domain), assembling the DISC. The DISC is an activation
platform that binds procaspase 8 through interactions
involving another type of motif called the death effec-
tor domain, which is present on both FADD and
the prodomain of procaspase 8. Procaspase 8 monomers
dimerize on the DISC and acquire catalytic activity.
These dimers can cleave neighboring dimers, creating
and releasing heterotetrameric active caspase 8, which
can initiate the caspase cascade by activating downstream
effector caspases. Frequently, caspase 8 cleaves the BH3-
only protein Bid, thereby activating the intrinsic death
pathway (Fig. 46.17).
The extrinsic pathway poses considerable risk for the
cell. Fas is constitutively present in the cell membrane
and can form at least transient trimers in the absence of
binding by its ligand. How do cells avoid the accidental
activation of apoptosis caused by chance binding of
procaspase 8 to naturally occurring transient Fas trimers?
Cells express a caspase-related protein called cFLIP
(FLICE-like inhibitory protein; FLICE is another name for
caspase 8) that looks very much like a catalytically dead

Unfolded FasR
binding domains

TARGET
FasR CD95 APO-1 CELL
8

8 8
A. Preligation
Monomers C. Release of active caspase 8
KILLER 8
CELL

Uncleaved caspase 8
Fas active on DISC
FADD
ligand Death domain 8

Death effector 8
Released active
domain caspase 8
Trimers DISC 8
(signaling
platform)
8

Procaspase 8

B. Ligand docked on a trimerized receptor Caspase 8

Molecular cleaves Bid Procaspases 3,6,7
scaffold forms 8 7
Bid 3 6

8
E. Activation of the D. Activation
intrinsic death of effector
8
pathway (see caspases 7
Clustering of Fig. 46.17) Active caspases 3,6,7
8
caspase 8
6 3
F. Death

FIGURE 46.18 EXTRINSIC CELL DEATH PATHWAY. The pathways shown are downstream of the Fas cell death receptor. A, Preligation.
B, Ligand docked on a trimerized receptor. C, Release of active caspase. D, Activation of effector caspases. E, Activation of the intrinsic death
pathway (Fig. 46.17). F. Death (see the text for a detailed description). DISC, death-inducing signaling complex; FADD, Fas-associated death
domain.
812 SECTION X n Cell Cycle
version of procaspase 8. When expressed at high levels,
cFLIP coassembles with procaspase 8 monomers creat-
ing an enzyme with altered activity that does not trigger
cell death. This role of cFLIP may be to dampen the Fas
response locally to ensure that the cascade does not get
activated by mistake. Another role of FLIP is to combine
with caspase 8 to ensure that TNF (tumor necrosis
factor ) signaling, which occurs in many immune cells,
promotes inflammation rather than killing cells by
necroptosis (see later).
Role of the Fas Death Receptor in Normal
and Diseased Cells
Fas is important for regulation of the immune system,
but also has a very unexpected role in cancer cells. Mice
with mutated Fas (the lpr mutation) or Fas ligand (the
gld mutation) accumulate excessive lymphocytes. In the
appropriate genetic background, these mice tend to
develop autoimmune disorders.
Fas is important in regulating the life span of activated
tissue T and B lymphocytes. Normally, T cells die within
a few days of their activation during an immune response.
Activation initiates the expression of Fas ligand on the T
cells themselves. This new Fas ligand interacts by an
unknown mechanism with Fas already on the cell surface,
causing the cell to commit apoptotic suicide. T-cell
activation also downregulates the expression of cFLIP,
thus permitting activated caspase 8 to more effectively
kill the cell. A similar mechanism (export of Fas and
Fas ligand to the surface of the same cell) is responsible
for some examples of p53-induced cell death and some
instances of cell death following exposure to chemo-
therapeutic agents.
These features of Fas might seem to make this system
useful in the treatment of cancer. Unfortunately this is
not the case, because Fas does more than signal to
promote cell death. It can also signal to several pathways
to promote cell survival and migration. In fact, Fas signal-
ing in cancer cells can actually promote tumor growth
and metastasis. This serves as an example of the com-
plexity of cell signaling networks.
Some tissues, like the lens of the eye and the testis,
avoid immune and inflammatory responses by express-
ing Fas ligand. Immune effector cells (which express
Fas on their surface) that enter these tissues encounter
Fas ligand and die by apoptosis. These tissues are known
as immune-privileged. Not surprisingly, certain tumor
cells subvert this strategy as protection against the
immune system. Melanoma cells expressing Fas ligand
establish tumors particularly efficiently. Some tumor
cells, especially those from colon and lung cancers, also
defend themselves against immune surveillance with
so-called decoy receptors. A secreted Fas decoy
receptor blocks Fas ligand on cytotoxic T cells so that
it cannot engage Fas on the surface of the tumor
cells. Other decoy receptors remain membrane bound
but do not signal cell death when they bind ligand
because their intracellular domains lack functional death
domains.
Linking Apoptosis to the Cell Cycle
by p53
No obligate link exists between particular cell-cycle
phases and apoptosis. Noncycling G0 cells can undergo
apoptosis, and cycling cells appear able to do so from
any cell-cycle phase. However, one link between apop-
tosis and the cell-cycle machinery is firmly established.
This involves the p53 tumor suppresser and DNA
damage.
The p53 transcription factor is one of the downstream
EFFECTORS of the DNA damage response pathway (see
Fig. 43.11). When cells sense DNA damage induced by
agents such as ionizing radiation, p53 levels rise dramati-
cally. When stabilized and activated by phosphorylation
(see Fig. 41.14) p53 upregulates the expression of a
number of genes, including the Cdk inhibitor p21, which
blocks the entry into the S and M phases. p53 also can
trigger an apoptotic response in instances in which the
DNA damage is too severe to repair (Fig. 46.19). This
tumor-suppressor protein is critical in the bodys defense
against cancer. Mutations in the p53 gene/protein are
found in approximately 50% of all human cancers.
A direct connection between p53 and apoptosis
was revealed by overexpressing the cloned p53 gene
in different cell types. In most cells, overexpression of
p53 arrests the cell cycle at the G1/S boundary.
However, ectopic expression of cloned p53 in certain
cancer-derived cell lines causes the cells to undergo
apoptosis.
The role of p53 in apoptosis was confirmed in trans-
genic mice lacking a functional p53 gene (p53 knockout
mice). These mice develop normally but are extremely
prone to cancer at a very young age. Thymocytes isolated
from p53 knockout mice are highly resistant to the
induction of apoptosis by ionizing radiation and other
agents that cause DNA breaks (Fig. 46.19B). However,
p53 is not involved in all types of apoptosis. For example,
the same thymocytes isolated from p53 knockout mice
show normal induction of apoptosis following exposure
to glucocorticoid hormone (Fig. 46.19).
p53 promotes apoptosis by functioning as a transcrip-
tional activator. It controls, among others, the well-
studied death-promoting genes Bax, Fas (CD95/APO-1),
and APAF-1. However, the key target gene is PUMA (p53
modulated upregulator of apoptosis), a BH3-only protein
that promotes apoptotic cell death by activating Bax and
Bak. PUMA knockout mice show defects in cell death
pathways that are essentially identical to those seen in
p53 knockout mice and not seen in mice that lack Bax,
Fas, or Apaf-1.

A. Glucocorticoid
hormonetreated
100
Cell viability (%)
Cell viability (%)
80
60
40
20
0 0
0 5 10 15 20 25
Time (h)
C. Control
FIGURE 46.19 LINK BETWEEN p53 AND DNA DAMAGE-
INDUCED APOPTOTIC DEATH. AB, Survival of thymocytes from
three strains of mice after exposure to glucocorticoids or irradiation.
Cell death was from apoptosis. The strains were as follows: wild-type
mice (yellow), heterozygous mice having one good copy of the p53
gene and one defective copy (red), and mice lacking any functional
copy of the p53 gene (blue). Thymocytes that lack p53 are resistant
to radiation-induced apoptosis but show normal induction of apopto-
sis following exposure to glucocorticoid hormone. CD, Induction of
p53 accumulation following radiation of the small intestine. Black
arrows indicate cells with increased levels of p53. Red arrows indicate
apoptotic cells. (AB, From Lowe SW, Schmitt EM, Smith SW, et al.
p53 is required for radiation-induced apoptosis in mouse thymocytes.
Nature. 1993;362:847849. CD, Courtesy John Hickman, Molecular
and Cellular Pharmacology Group, University of Manchester, United
Kingdom.)
Other Types of Programmed Cell Death
The terms apoptosis and programmed cell death are
sometimes viewed as synonymous. However, a number
of specific examples of programmed cell death have
been described that lack the features that classically
define apoptosis.
Inflammatory Cell Death: Pyroptosis
and Necroptosis
One of the defining features of apoptosis is that dying
cells disappear without causing an inflammatory
response. However, in certain instances, it is protective
for the body to have cell death be accompanied by
CHAPTER 46 n Programmed Cell Death 813
B. Irradiated inflammation as this can trigger an immune response in
the early phases of infection.
p53 Pyroptosis (proinflammatory programmed cell
100 / death) occurs when signaling by a variety of cytoplasmic
80
receptors in response to pathogen infection leads to the
60 formation of the inflammasome, a cytosolic complex
40
p53 that contains numerous caspase 1 activation sites. Pyrin
+/
20 +/+
domains (PYDs) in these receptors are linked to caspase
recruitment domains (CARDs), which in turn recruit
0 5 10 15 20 25 caspases 1, 4, and 5 (1 and 11 in mice). Despite being
Time (h) the first caspase to be described, caspase 1 was the last
to be shown to be involved in a programmed cell death
D. Irradiated response.
The function of caspase 1 is to produce interleukin-1
(IL-1) and interleukin-18 (IL-18) by proteolytic process-
ing of their precursors. These proteins are secreted
together with caspase 1 by an unconventional pathway
that is still being investigated and may involve cell lysis.
IL-1 is a proinflammatory cytokine that triggers the
fever response by acting on the hypothalamus. It also
promotes the proliferation of immune cells, among other
activities. IL-18 promotes increased immune cell recruit-
ment and activation, as well as modifying local cells to
facilitate adaptive immune responses. This proinflamma-
tory signaling facilitates a ramping up of the immune
response to the pathogen.
How pyroptosis leads to cell death is still being inves-
tigated, but the morphology of the dying cells resembles
that seen in necrosis. As a result, the internal components
of the cell are released into the extracellular space,
provoking a strong inflammatory response (as in necro-
sis, above, and necroptosis, below).
Necroptosis (programmed necrosis) is a cell death
pathway that may function as a backup when the extrinsic
pathway of apoptosis is not functioning. Activation of the
CD95 (Fas) and TNFR1 (tumor necrosis factor receptor
1) cell-surface receptors can have three possible out-
comes. TNFR1 can signal via complex I (which contains
protective IAP proteins) to activate the canonical NF-B
pathway to promote cell survival (Fig. 46.1). Alternatively
TNFR1 can activate receptor interacting protein kinases 1
and 3 (RIPK1/RIPK3) and signal to form complex II (con-
taining FADD, TRADD [tumor necrosis factor receptor-
associated death domain], caspase 8, and cFLIP). When
caspase 8 is associated with cFLIP, it inactivates RIPK3
and cells survive. Complex II activates necroptosis if
either caspase 8 or cFLIP is missing or inactivated and
if RIPK1/RIPK3 are active. RIPK1/RIPK3 activity leads
to permeabilization of the cell membrane, cell lysis and
death. As in other forms of necrosis, the released cellular
contents cause a local inflammatory response.
Necroptosis is thought to be a backup pathway for
apoptosis during viral infection. Mouse kidney cells
infected with a virus that encodes a caspase 8 inhibitor
proceed down the necroptosis death pathway. Removal
of the gene from the virus restores apoptosis. Mice
814 SECTION X n Cell Cycle

lacking RIPK3 show increased susceptibility to viral

infection. Murine cytomegalovirus has proteins that help
it to evade both apoptosis and necroptosis, supporting Primary
lesion
this argument.

Autophagic Death
Autophagy is a catabolic, energy producing process that
allows cells to survive in adverse conditions by recycling
amino acids liberated by degradation of cellular proteins
and organelles (see Fig. 23.7). Autophagy can be acti-
vated by a wide range of stimuli, including nutrient and
oxidative stress, accumulation of unfolded proteins,
intracellular bacterial infection, and oncogenic stress. In
addition to being a response to starvation, autophagy can
also participate in antiviral responses by participating in
antigen presentation by immune cells.
Connections between autophagy and cell death are
complex and debated. In certain cases during develop-
ment, autophagic pathways can lead directly to cell
death. These dying cells lack chromatin condensation
and exhibit extreme vacuolization of the cytoplasm. In
some cells, autophagy can regulate apoptosis. For
example, degradation of proapoptotic BH3-only proteins
by autophagy provides a mechanism protecting cells
against apoptosis. Alternatively, degradation of protec-
tive proteins can cause autophagy to actively promote
apoptotic cell death.
Connections between autophagy and cancer are
complex. It has been argued that by promoting cell
survival under adverse conditions, autophagy might help
promote the establishment of cancers particularly in
avascular areas and by helping cancer cells to survive
chemotherapy. However, mice heterozygous for Beclin1
(a scaffolding protein that functions early in assembly of
the phagophore membrane; see Fig. 23.7) develop
cancers, suggesting that Beclin-1 is a tumor suppressor
and that autophagy may protect against tumorigenesis.
Interestingly, Beclin-1 has a BH3 domain, and although
it is not a canonical BH3-only proapoptotic protein, its
function is negatively regulated by Bcl-2. Also, autophagy
is activated in oncogene-induced senescence and inhibit-
ing autophagy delays senescence. Thus autophagy might
prevent cancer formation by helping damaged cells to
become senescent.
Importance of Programmed Cell Death in
Human Disease
Why have studies of programmed cell death so caught
the scientific eye? One likely answer is that cell death is
a point of intersection between cell signaling pathways,
cell structure, the cell cycle, and, of course, human
disease. This chapter has mentioned the roles that aber-
rations in programmed cell death play in the etiology
of autoimmunity, AIDS, and cancer. Cell death is firmly
established as a key factor in neurodegenerative diseases,
Penumbra: zone of apoptotic Primary focus
death starting within 24 hours of necrotic death
of the initial lesion
FIGURE 46.20 BRAIN DAMAGE IN STROKE. Secondary pro-
grammed cell death caused by oxygen deprivation in the penumbra
greatly increases the size of the affected area of the brain in stroke.
such as Huntington disease and Alzheimer disease, as
well as in myocardial infarction and stroke (Fig. 46.20).
At a practical level, the realization that many successful
chemotherapeutic agents act by inducing cancer cells
to undergo apoptosis motivated searches for newer
and better drugs that elicit this response. The generally
disappointing outcome of those studies may largely be
because other pathways (eg, necroptosis) kick in when
apoptosis is inhibited. Alternatively, as more is learned
about mechanisms of necroptosis and pyroptosis, these
alternative types of programmed death become attractive
therapeutic targets, as their induction may not only kill
the target cells, but also provoke a localized immune
response that might attack cancer cells or pathogens.
With such important practical problems to be solved, pro-
grammed cell death will continue to occupy a prominent
position in cell biology research over the coming years.
ACKNOWLEDGMENTS
We thank Charles Earnshaw, Scott Kaufmann, Luis
Miguel Martins, and Andrew Thorburn for their sugges-
tions on revisions to this chapter.
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