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.
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 platelet-derived
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)
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
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.
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