Instructor’s Manual
to accompany
The Cell: A Molecular Approach, Fifth Edition
Chapter 17 Cell Death and Cell Renewal
Chapter Outline and Summary
PROGRAMMED CELL DEATH
The Events of Apoptosis: Programmed cell death plays a key role in both the
maintenance of adult tissues and embryonic development. In contrast to the accidental
death of cells from an acute injury, programmed cell death takes place by the active
process of apoptosis. Apoptotic cells and cell fragments are then efficiently removed by
phagocytosis. Genes responsible for the regulation and execution of apoptosis were
initially identified by genetic analysis of C. elegans.
See Website Animation 17.1: Apoptosis: During apoptosis, chromosomal DNA is
usually fragmented, the chromatin condenses, the nucleus breaks up, and the cell
shrinks and breaks into apoptotic bodies.
Key Experiment: Identification of Genes Required for Programmed Cell Death
Caspases: The Executioners of Apoptosis: The caspases are a family of proteases that
are the effectors of apoptosis. Caspases are classified as either initiator or effector
caspases, and both function in a cascade leading to cell death. In mammalian cells, the
major initiator caspase is activated in a complex called the apoptosome, which also
requires cytochrome c released from mitochondria.
Central Regulators of Apoptosis: the Bcl-2 Family: Members of the Bcl-2 family are
central regulators of caspase activation and apoptosis. Some members of the Bcl-2 family
function to inhibit apoptosis (antiapoptotic) whereas others act to promote apoptosis
(proapoptotic). Signals that control programmed cell death alter the balance between
proapoptotic and antiapoptotic Bcl-2 family members, which regulate one another. In
mammalian cells, proapoptotic Bcl-2 family members act at mitochondria, where they
promote the release of cytochrome c, leading to caspase activation. Caspases are also
regulated directly by inhibitory IAP proteins.
See Website Animation 17.2: The Mitochondrial Pathway of Apoptosis: Many forms
of cell stress activate the intrinsic pathway of apoptosis—a pathway that leads to the
release of cytochrome c from mitochondria, the activation of caspase-9, and the
subsequent death of the cell.
Signaling Pathways that Regulate Apoptosis: A variety of signaling pathways regulate
apoptosis by controlling the expression or activity of proapoptotic members of the Bcl-2
family. These pathways include DNA damage-induced activation of the tumor suppressor
p53, growth factor-stimulated activation of PI 3-kinase/Akt signaling, and activation of
death receptors by polypeptides that induce programmed cell death.
Alternative Pathways of Programmed Cell Death: Autophagy and regulated necrosis
provide alternatives to apoptosis for induction of programmed cell death.
STEM CELLS AND THE MAINTENANCE OF ADULT TISSUES
Proliferation of Differentiated Cells: Most cells in adult animals are arrested in the G0
stage of the cell cycle. A few types of differentiated cells, including skin fibroblasts,
endothelial cells, smooth muscle cells, and liver cells are able to resume proliferation as
required to replace cells that have been lost because of injury or cell death.
Stem Cells: Most differentiated cells do not themselves proliferate but can be replaced
via the proliferation of stem cells. Stem cells divide to produce one daughter cell that
remains a stem cell and another that divides and differentiates. Stem cells have been
identified in a wide variety of adult tissues, including the hematopoietic system, skin,
intestine, skeletal muscle, brain, and heart.
Medical Applications of Adult Stem Cells: The ability of stem cells to repair damaged
tissue suggests their potential use in clinical medicine. Adult stem cells are used to repair
damage to the hematopoietic system in hematopoietic stem cell transplantation, and
epidermal stem cells can be used for skin grafts. However, clinical applications of adult
stem cells are limited by difficulties in isolating and culturing these cells.
EMBRYONIC STEM CELLS AND THERAPEUTIC CLONING
Embryonic Stem Cells: Embryonic stem cells are cultured from early embryos. They can
be readily grown in the undifferentiated state in culture while retaining the ability to
differentiate into a wide variety of cell types, so they may offer considerable advantages
over adult stem cells for many clinical applications.
Key Experiment: Culture of Embryonic Stem Cells
Somatic Cell Nuclear Transfer: Mammals have been cloned by somatic cell nuclear
transfer in which the nucleus of an adult somatic cell is transplanted into an enucleated
egg. This opens the possibility of therapeutic cloning in which embryonic stem cells
would be derived from a cloned embryo and used for transplantation therapy of the donor
of the adult nucleus. Although many obstacles need to be overcome, the possibility of
therapeutic cloning holds great promise for the development of new treatments for a
variety of devastating diseases.
Induced Pluripotent Stem Cells: Adult somatic cells can be converted to pluripotent
stem cells in culture by four key transcription factors, potentially providing an alternative
to embryonic stem cells for transplantation therapy.
Lecture Notes
Introduction
 Cell death and cell proliferation are balanced throughout the life of multicellular
organisms.
 Animal development involves not only cell proliferation and differentiation but
also cell death.
 Most cell deaths occur by a normal physiological process of programmed cell
death.
 In adult organisms, cell death must be balanced by cell renewal, and most tissues
contain stem cells that are able to replace cells that have been lost.
Programmed Cell Death
 Programmed cell death is carefully regulated.
 In adults, it balances cell proliferation and maintains constant cell numbers, and
eliminates damaged and potentially dangerous cells.
 During development, programmed cell death plays a key role by eliminating
unwanted cells from a variety of tissues.
 The Events of Apoptosis
o Necrosis = accidental cell death; apoptosis = programmed cell death.
o Apoptosis is an active process of programmed cell death, characterized by
cleavage and fragmentation of chromosomal DNA, chromatin condensation,
and fragmentation of both the nucleus and the cell. (Figure 17.1)
o Apoptotic cells and cell fragments are efficiently recognized and phagocytosed
by both macrophages and neighboring cells; cells that die by apoptosis are
rapidly removed from tissues. (Figure 17.2) Necrotic cells swell and lyse, the
contents are released into the extracellular space and cause inflammation.
o Apoptotic cells express “eat me” signals such as phosphatidylserine. In normal
cells, phosphatidylserine is restricted to the inner leaflet of the plasma
membrane.
o Three genes have been identified that play key roles in regulating and
executing apoptosis: ced-3, ced-4, and ced-9. (Figure 17.3 and Key
Experiment using C. elegans)
o These genes are the central regulators and effectors of apoptosis and are highly
conserved in evolution.
 Caspases: The Executioners of Apoptosis
o Ced-3 is the prototype of a family of proteases, known as caspases. They have
cysteine (C) residues at their active sites and cleave after aspartic acid (Asp)
residues in their substrate proteins.
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o Caspases are the ultimate effectors of programmed cell death, bringing about
the events of apoptosis by cleaving nearly 100 different cell target proteins.
(Figure 17.4)
o Ced-4 and its mammalian homolog (Apaf-1) bind to caspases and promote
their activation. In mammalian cells, caspase-9 is activated by binding to
Apaf-1 in a protein complex called the apoptosome. Cytochrome c is also
required, which is released from mitochondria. (Figure 17.5)
Central Regulators of Apoptosis: The Bcl-2 Family
o ced-9 in C. elegans is closely related to a mammalian gene called bcl-2, first
identified as an oncogene. Bcl-2 inhibits apoptosis. Cancer cells are unable to
undergo apoptosis.
o Mammalian cells encode about 20 proteins related to Bcl-2, in three functional
groups. Some inhibit apoptosis, while others induce caspase activation.
(Figure 17.6)
o The fate of the cell is determined by the balance of activity of proapoptotic
and antiapoptotic Bcl-2 family members. (Figure 17.7)
o In mammalian cells, members of the Bcl-2 family act at mitochondria, which
play a central role in controlling programmed cell death. (Figure 17.8)
o Caspases are also regulated by a family of proteins called the IAP (inhibitor of
apoptosis). They suppress apoptosis by either inhibiting caspase activity or by
targeting caspases for ubiquitination and degradation in the proteasome.
(Figure 17.9)
Signaling Pathways that Regulate Apoptosis
o Regulation of programmed cell death is mediated by the integrated activity of
a variety of signaling pathways, some acting to induce cell death and others
acting to promote cell survival.
o Many forms of cell stress, such as DNA damage, viral infection, and growth
factor deprivation can trigger programmed cell death.
o A major pathway leading to cell cycle arrest in response to DNA damage is
mediated by the transcription factor p53. Activation of p53 by DNA damage
can also lead to apoptosis. (Figure 17.10)
o A major intracellular signaling pathway that promotes cell survival is initiated
by the enzyme PI 3-kinase, which phosphorylates the membrane phospholipid
PIP2 to form PIP3, which activates Akt. Akt then phosphorylates a number of
proteins that regulate apoptosis. (Figure 17.11)
o Polypeptides in the tumor necrosis factor (TNF) family signal cell death by
activating cell surface receptors. These receptors directly activate a distinct
initiator caspase, caspase-8. (Figure 17.12)
Alternative Pathways of Programmed Cell Death
o Programmed cell death can also occur by alternative, non-apoptotic
mechanisms such as autophagy.
o In normal cells, autophagy provides a mechanism for the gradual turnover of
the cell’s components by the uptake of proteins or organelles into vesicles that
fuse with lysosomes.
o Autophagy can be an alternative to apoptosis as a pathway of cell death. It
does not require caspases, and may be activated by cellular stress and provide
an alternative to apoptosis when apoptosis is blocked.
o Some forms of necrosis can be a programmed cellular response to stimuli such
as infection or DNA damage, and may provide an alternative pathway of cell
death if apoptosis does not occur.
Stem Cells and the Maintenance of Adult Tissues
 Early development is characterized by the rapid proliferation of embryonic cells,
which then differentiate to form the specialized cells of adult tissues and organs.
 In order to maintain a constant number of cells in adult tissues and organs, cell
death must be balanced by cell proliferation.
 Proliferation of Differentiated Cells
o Most types of differentiated cells in adult animals are no longer capable of
proliferation. If these cells are lost they are replaced by proliferation of cells
derived from self-renewing stem cells.
o Some types of differentiated cells retain the ability to proliferate as needed, to
repair damaged tissue throughout the life of the organism.
o Fibroblasts in connective tissue can proliferate quickly in response to plateletderived growth factor (PDGF) released at the site of a wound. (Figure 17.13)
o The endothelial cells that line blood vessels are another type of fully
differentiated cell that remains capable of proliferation to form new blood
vessels for repair and regrowth of damaged tissue. (Figure 17.14)
o The epithelial cells of some internal organs, such as the liver and pancreas, are
also able to proliferate to replace damaged tissue. (Figure 17.16)
 Stem Cells
o Most fully differentiated cells in adult animals are no longer capable of cell
division.
o Stem cells are less differentiated, self-renewing cells present in most adult
tissues. They retain the capacity to proliferate and replace differentiated cells
throughout the lifetime of an animal.
o Stem cells divide to produce one daughter cell that remains a stem cell and
one that divides and differentiates. (Figure 17.17)
o Many types of cells have short life spans and must be continually replaced by
proliferation of stem cells: blood cells, sperm, epithelial cells of the skin and
lining the digestive tract.
o Hematopoietic stem cells are well-characterized. They give rise to several
distinct types of blood cells with specialized functions: erythrocytes,
granulocytes, macrophages, platelets, and lymphocytes. (Figure 17.18)
o Epithelial cells that line the intestines live only a few days before they die by
apoptosis. New cells are derived from the continuous but slow division of
stem cells at the bottom of intestinal crypts. (Figure 17.19)
o Skin and hair are also renewed by stem cells. The epidermis, hair follicles, and
sebaceous glands are all maintained by their own stem cells. (Figure 17.20)
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o Stem cells also play a role in the repair of damaged tissue. Skeletal muscle is
normally has little cell turnover, but it can regenerate rapidly in response to
injury or exercise. Regeneration is mediated by proliferation of satellite
cells—the stem cells of adult muscle. (Figure 17.21)
o Stem cells have also been identified in many other adult tissues, including the
brain, retina, heart, lung, kidney, liver, and pancreas, and it is possible that
most—if not all—tissues contain stem cells.
o Stem cells reside in distinct microenvironments or niches which provide the
environmental signals that maintain stem cells throughout life and control the
balance between self-renewal and differentiation.
Medical Applications of Adult Stem Cells
o The ability of adult stem cells to repair damaged tissue clearly suggests their
potential utility in clinical medicine.
o A bone marrow transplantation is a clinical procedure in which transplantation
of bone marrow stem cells is used in the treatment of cancer and diseases of
the hematopoietic system. (Figure 17.22)
o Epithelial stem cells have clinical application in the form of skin grafts that
are used to treat patients with burns, wounds, and ulcers.
Embryonic Stem Cells and Therapeutic Cloning
 Embryonic stem cells can be grown indefinitely as pure stem cell populations that
have pluripotency - the capacity to develop into all of the different types of cells in
adult tissues.
 There is enormous interest in embryonic stem cells from the standpoints of both
basic science and clinical applications.
 Embryonic Stem Cells
o Embryonic stem cells were first cultured from mouse embryos in 1981.
(Figure 17.23)
o Mouse embryonic stem cells have been an important experimental tool in cell
biology because they can be used to introduce altered genes into mice and they
provide an outstanding model system for studying the molecular and cellular
events associated with cell differentiation.
o Human embryonic stem cell lines were first established in 1998. Clinical
transplantation therapies based on embryonic stem cells may provide the best
hope for treatment of diseases such as Parkinson’s, Alzheimer’s, diabetes, and
spinal cord injuries.
o Mouse embryonic stem cells are grown in the presence of growth factor LIF,
which signals through the JAK/STAT pathway and is required to maintain
these cells in their undifferentiated state. If LIF is removed, the cells aggregate
and differentiate. (Figure 17.24)
o Importantly, embryonic stem cells can be directed to differentiate along
specific pathways by the addition of appropriate growth factors to the culture
medium.
 Somatic Cell Nuclear Transfer
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o The isolation of human embryonic stem cells in 1998 followed the first
demonstration that the nucleus of an adult mammalian cell could give rise to a
viable cloned animal when Dolly the sheep was cloned. (Figure 17.25)
o Somatic cell nuclear transfer is the basic procedure of animal cloning in which
the nucleus of an adult somatic cell is transferred to an enucleated egg. This
type of cloning in mammals is a difficult and inefficient process.
o In therapeutic cloning, a nucleus from an adult human cell might be
transferred to an enucleated egg, which would then be used to produce an
early embryo in culture. The resulting embryo could produce differentiated
cells for transplantation therapy. This would bypass the problem of tissue
rejection. (Figure 17.26)
o Problems to be overcome include the low efficiency of generating embryos by
somatic cell nuclear transfer; ethical concerns with respect to the possibility of
cloning human beings (reproductive cloning), and with respect to the
destruction of embryos.
Induced Pluripotent Stem Cells
o These technical and ethical difficulties may be overcome by using induced
pluripotent stem cells—reprogramming somatic cells to resemble embryonic
stem cells.
o The action of only four key transcription factors is sufficient to reprogram
adult mouse somatic cells. (Figure 17.27)
o Adult human fibroblasts can be reprogrammed to pluripotency by a similar
procedure. Although problems remain, induced pluripotent stem cells may
someday be used for patient-specific transplantation therapy.
Key Terms
Akt
apoptosis
apoptosome
autophagy
Bcl-2
bone marrow transplantation
caspase
embryonic stem cell
hematopoietic stem cell transplantation
IAP
induced pluripotent stem cell
necrosis
niche
p53
PI 3-kinase
pluripotency
programmed cell death
reproductive cloning
somatic cell nuclear transfer
stem cell
therapeutic cloning
tumor necrosis factor (TNF)
End-of-Chapter Questions and Answers
1. Why is cell death via apoptosis more advantageous to multicellular organisms
than cell death via acute injury?
Answer: Apoptotic cells are efficiently removed from tissues by phagocytosis, whereas
cells that die by acute injury release their contents into the extracellular space and cause
inflammation.
2. What molecular mechanisms regulate caspase activity?
Answer: Caspases are synthesized as long inactive precursors that are activated in
complexes (e.g., the apoptosome) or converted to active enzymes by proteolytic cleavage.
In addition, cells contain IAPs that associate with caspases and inhibit their activity.
3. You have expressed mutants of nuclear lamins in human fibroblasts. The Asp
residue in the caspase cleavage site has been mutated to Glu in these lamins. How
would these mutant lamins affect the progression of apoptosis?
Answer: The cleavage of lamins by caspases is required for nuclear fragmentation during
apoptosis. The mutated lamins will not be cleaved by caspases, so their expression will
block nuclear fragmentation.
4. How do Bcl-2 family proteins regulate apoptosis in mammalian cells?
Answer: Proapoptotic multidomain members of the Bcl-2 family induce apoptosis by
promoting the release of cytochrome c from mitochondria, which leads to caspase
activation. The activity of the proapoptotic multidomain proteins is regulated by
antiapoptic and BH3-only members of the Bcl-2 family.
5. How does p53 activation in response to DNA damage affect cell cycle progression
and cell survival?
Answer: Activation of p53 in response to DNA damage leads to the expression of its
target genes, which include the Cdk inhibitor p21 and the BH3-only Bcl-2 family
members PUMA and Noxa. p21 induces cell cycle arrest and the BH3-only Bcl-2 family
members induce apoptosis.
6. You have constructed a Bad mutant in which the Akt phosphorylation site has
been mutated such that Akt no longer phosphorylates it. How would expression of
this mutant affect cell survival?
Answer: The mutant Bad would no longer be maintained in an inactive state by 14-3-3
protein, so it will act to induce apoptosis.
7. How would expression of siRNA targeted against 14-3-3 proteins affect apoptosis?
Answer: 14-3-3 proteins sequester proapoptotic proteins, such as Bad and FOXO
transcription factors, in an inactive state. Cells expressing siRNA against 14-3-3 proteins
will therefore have an increased rate of apoptosis.
8. You are considering treatment of a leukemic patient with TNF. Upon further
analysis you determine that the leukemic cells have an inactivating mutation of
caspase-8. Will treatment with TNF be an effective therapy for this patient?
Answer: Caspase-8 is the initiator caspase downstream of TNF receptors, so cells with
inactive caspase-8 will not undergo apoptosis upon treatment with TNF. Thus TNF
therapy would not be effective for this patient.
9. How would siRNA against Ced-3 affect the development of C. elegans?
Answer: Ced-3 is the only caspase in C. elegans. Mutating it leads to the survival of all
the cells that would normally die by apoptosis during development, and RNAi against
Ced-3 would have the same effect.
10. You have isolated a polypeptide from a toxic plant, which localizes to
mitochondria after endocytosis by mammalian cells. The polypeptide aggregates
and forms large channels in the mitochondrial outer membrane, releasing proteins
from the intermembrane space into the cytoplasm. How will treatment with this
polypeptide affect mammalian cells in culture?
Answer: The polypeptide will lead to the release of cytochrome c from mitochondria and
induce apoptosis of treated cells.
11. Many adult tissues contain terminally differentiated cells that are incapable of
proliferation. However, these tissues can regenerate following damage. What gives
these tissues their regenerative capabilities?
Answer: These tissues contain stem cells that retain the ability to proliferate and replace
differentiated cells.
12. What is the critical property of stem cells?
Answer: The critical characteristic of stem cells is their capacity for self-renewal. They
divide to produce one daughter cell that remains a stem cell and one that divides and
differentiates.
13. What are the potential advantages of embryonic stem cells as compared to adult
stem cells for therapeutic applications?
Answer: Embryonic stem cells are easier to isolate and culture and are capable of giving
rise to all of the differentiated cell types in an adult organism.