Programmed Cell Death 1
Programmed Cell Death:
Parallels Between Neurons and Somatic Cells
Jason Pitt
jp101@evansville.edu
Psyc 490
Instructor and Grader: Dr. Lakey
November 6, 2006Personal Relevance Preface
Ultimately, my career goal is to teach biology or neuroscience at the university level
Programmed Cell Death 2
and conduct research in that field. This path will be one of lifelong learning. Working as a
professor will provide me with the opportunity to remain up-to-date on the subjects in which I
am interested, and performing research will allow me to remain on the cutting edge. Requisite
to this is obtaining my PhD. Therefore, after graduation, I will be attending graduate school.
So why write a paper on cell death, especially neuronal cell death? Well, cell death is
a part of many important processes, from normal development to cancer prevention.
Misregulation of cell death can have disastrous effects on an organism (e.g., tumorogenesis).
Therefore, understanding cell death has important implications in many aspects of research.
The reason my paper will focus on neuronal cell death in the end is because of my fascination
with the brain. As I see neuroscience as a definite possibility in my future, knowledge about
how neurons kill themselves could be very important.
The main objective of my paper is to outline the major molecular pathways involved
with programmed cell death in both “normal” somatic cells and neurons. There are major
differences between somatic cells and neurons, most notably, “normal” cells are made to die
eventually, while neurons have the potential to live as long as the organism. An
understanding of neuronal cell death could also have important implications in understanding
neurodegenerative disorders.
Table of Contents
Introduction...............................................................................................................
Programmed Cell Death 3
....................4
Different Types of Cell
Death...............................................................................................5
Why Do Cells Kill
Themselves?..........................................................................................6
The Cytology of Neurons and “Normal” Somatic
Cells.....................................................9
Molecular Basis of
Apoptosis....................................................................................................11
What Keeps a “Normal” Cell
Alive?.................................................................................12
How Do Cells Kill
Themselves?........................................................................................13
What Happens When Cells Fail to
Die?...........................................................................22
Neuronal Specific Cell
Death....................................................................................................23
What Keeps Neurons
Alive?.............................................................................................23
How Do Neurons Kill
Programmed Cell Death 4
Themselves?..................................................................................26
Other Players in Programmed Cell
Death..............................................................................28
Apoptosis Summary: A Lesson From
Worms.........................................................................30
Conclusion.................................................................................................................
..................31
References.................................................................................................................
...................33Introduction
When you look at your hand you see five fingers, but sometimes it is what you do
not see that is important. What you do not see are the spaces that give you five separate
fingers, instead of one fin. Missing from your hand are large groups of cells that were
specifically targeted for death while you were still developing, thereby sculpting away the cells
around your soon-to-be finger cells to give you five digits. This is just one of the many ways
that highly-controlled programmed cell death helps you function properly.
The control of cell death is vital – not enough cell death can allow cells to
accumulate unnaturally, while too much can cause the destruction of crucial body parts.
Cancer is a great example of a negative consequence of insufficient cell death. Cancer cells
escape cell cycle control through a series of mutations, and are thus highly abnormal. Each
mutation a cell suffers has the potential to change the physiology for better or worse. If
Programmed Cell Death 5
enough mutations accumulate to allow the cell to escape cell cycle control and avoid cell
death, cancers form. For this reason, the cell has many safeguards to protect its genomic
integrity.
Just as aberrant regulation of cell death can increase the cell's longevity, it can
also create death-prone cells. Excessive cell death can lead to disastrous effects, including
senility, improper motor functioning, and even death. In the nervous system, there is not an
abundance of extra cells to replace dead neurons. Therefore, neurons have the need to live
as long as the organism in which they exist. Diseases that target neurons for death can be
extremely devastating, as the cells that are dead will not be replaced.
However, neurons are not simply instructed to live forever when they are made. In
normal human nervous system development, many more neurons are created than are
necessary. This insures that every place that requires innervation will receive it. Humans
have a maximum number of neurons during week 28 of gestation; by birth the number has
decreased by about 70% (Rabinowicz, de Courten-Myers, Petetot, Xi, & de los Reyes, 1996).
The excess neurons die from a lack of neurotrophins, producing a less “cluttered” brain that
sends signals more efficiently.
Different Types of Cell Death
Although there is a need to control cell death, there are times when cell death is
not controlled, but rather proceeds in an unorganized fashion due to some sort of physical
insult to the cell (i.e., any condition that causes the cellular membrane to rupture).
Programmed Cell Death 6
Unprogrammed cell death can occur from excessive heat or cold, physical trauma, such as a
cut, or build-up of chemicals to toxic levels (Bhatia, 2003). In unprogrammed cell death, the
cell ruptures, spilling its contents into the extracellular environment. This type of cell death
has been termed necrosis, and while it is involved in many important biological functions (e.g.,
cancer treatment, normal development, etc.), it is not as well understood as programmed cell
death (apoptosis) and will therefore not be discussed in further detail (Zong & Thompson,
2006). Table 1 provides an adequate summary of the differences between the two types of
cell death.
Apoptosis is called programmed cell death (PCD) because the cell actually carries
Table 1: Necrosis vs. Apoptosis (from Sastry & Rao,
2000.)
out a defined sequence of events to kill itself. The sequence of molecular events in apoptosis
are as follows: a death signal is received; apoptotic proteins are activated, and these both
multiply the death response through a positive feedback loop as well as carry out apoptosis by
directly degrading cellular components or activating other hydrolytic proteins (Campbell &
Reece, 2002). Apoptosis is essentially a cell cutting up all of its components, and thereby
Programmed Cell Death 7
destroying any harmful substances before breaking apart. This gives apoptosis a major
advantage over necrosis in multicellular organisms. After necrotic cell death, the contents of
a cell are spilled out and can cause an inflammatory response, whereas apoptotic cell death
poses no threat of damage to neighboring cells (Alberts et al., 2004).
Another putative type of programmed cell death is called autophagy (from the Latin
“self eating”). In autophagy, digestive components called autophagosomes form to breakdown
old, damaged molecules/organelles into their constituent components for reuse (Yue, Jin,
Yang, Levine, & Heintz, 2003). Autophagy activation in response to starvation and cellular
stress, appears to serve a cytoprotective function (Yue, et al., 2003). Despite the role of
autophagy in protecting the cell, many groups still classify it as a type of programmed cell
death (Yu, et al., 2004; Boya, et al., 2005; Qu, et al., 2003). The role of autophagy in cell
death is far from clear. It is clear that autophagy is carried out by cells to prolong their lives
in situations where nutrients are scarce, but evidence also indicates a possible role of
autophagy in cell death. As the functions of autophagy are still being debated, it will also not
be focused on in subsequent sections.
Why Do Cells Kill Themselves?
Apoptosis is a highly regulated process and requires activation in order to proceed.
All of the apoptotic-mediating proteins are present in the cell during the cell's life cycle in an
inactive form. These are activated or deactivated in the presence of certain signals.
Proapoptotic signals can be intracellularly or extracellularly derived, and can initiate
Programmed Cell Death 8
apoptosis in a few different ways. Intracellular signals are usually protein signaling cascades
initiated by some cellular aberration (e.g., activation of the protein p53 initiates apoptosis
when the cellular genome becomes irregular). An external inducer of cell death is an
environmental insult. This includes radiation, cytotoxic chemicals, and removal of survival
factors (Zhang, Dimtehev, Dritschilo, & Jung, 2001; Alberts et al., 2004).
Highly damaged cells are prime targets for apoptosis. The genome of a cell
controls its physiology, and must therefore be free from damage or mutations in order for the
cell to function properly. Mutations can be disastrous, as they can obliterate vital protein
functions, which may lead to cell death, or immortality – cancer. For this reason, there is a
system within the cell to detect DNA damage, halt cell cycle progression, and if the damage
remains unrepaired cause the cell the undergo apoptosis. This is a necessary system in
multicellular organisms, as a cancerous cell grows uncontrollably and will begin to take away
space and nutrients from non-cancerous cells. Cells with extensive mitochondrial damage will
also initiate apoptosis. Unlike DNA damage, which causes cell death indirectly through
another system, compromised mitochondrial membrane integrity directly activates the
apoptotic pathway.
Apoptosis is involved in many different biological processes. One example is the
formation of structures during development. As stated earlier, apoptosis is responsible for
“sculpting” fingers out of the ball of prospective hand cells, and it also is responsible for
removing the tails of tadpoles as they metamorphose into frogs (Alberts et al., 2004).
Programmed Cell Death 9
Formation of gross body structures is an easily seen outcome of apoptosis, but there are also
many unseen systems that are “sculpted” by apoptosis.
During prenatal development, the lungs do not serve as the major hub of gas
exchange as in adults. There is a major rearrangement in the lungs necessary for proper
functioning. As the fetal lungs contain structures adult lungs do not need, and vice versa,
apoptosis is used to clear away the fetal structures, as well as remove the excess stem cells
used to produce the adult lung formations (Del Riccio, van Tuyl, & Post, 2004). Kresch,
Christian, Wu, and Hussain (1998) found a dramatic increase of apoptotic cells in lungs at
postnatal day one compared to prenatal tissue. In addition, the levels of apoptosis quickly
decreased in subsequent days. These results show that the useless fetal structures are
quickly removed by apoptosis to transition the lungs into their adult form.
Programmed cell death is also critical for immune system development.
Lymphocytes, leukocytes mediating the immune response, only survive if they are functional.
Lymphocytes that are not active (i.e. do not contain B cell antigen receptors or T cell antigen
receptors) will not be able to avoid cell death, and lymphocytes that are too active (i.e. react
strongly with self antigens to trigger cell death) will bring about their own death (Opferman &
Korsmeyer, 2003). The avoidance of apoptosis by lymphocytes can only be achieved if they
are functional, thus apoptosis ensures that the immune system will contain functional cells.
Another system shaped by apoptosis is the nervous system. As stated earlier, well
over half of the neurons made will be destroyed before birth (Rabinowicz et al., 1996). The
Programmed Cell Death 10
exact mechanisms of apoptosis in neural cells are outlined in later sections. Differences in
apoptotic function should be expected between neurons and “normal” somatic cells given the
differences between the cells. As we will see, the physiology of these two cell types are
different, which leads to very different behaviors.
The Cytology of Neurons and “Normal” Somatic Cells
A common theme in biology is that form determines function. Following this, one
would expect neurons to be vastly different from a somatic cell, as they have very different
functions. Neurons are highly elongated and are capable of producing rapid changes in
membrane potential in a certain direction, as they are responsible for signaling between all
areas of the body. Because there are so many different functions of somatic cells, there is
also a variety of cell types within this group. To give some idea of how diverse cells are
within the body, red blood cells are anucleated and short-lived, whereas stem cells can
theoretically divide forever. Nevertheless, we can make some generalizations about the
similarities and differences between the morphologies of neuronal and somatic cells.
There are some basic cellular components that must be present in all viable cells
capable of division. Each cell is surrounded by a plasma membrane, and within each cell is a
nucleus, an endomembrane system, and a system for providing nutrients and removing waste.
The plasma membrane is a lipid bilayer that contains the intracellular contents. Within the
nucleus is the genome of each cell. The nucleus is the site of many important processes that
promote cell survival, including RNA transcription and DNA replication. The endomembrane
Programmed Cell Death 11
system consists of the endoplasmic reticulum, the Golgi apparatus, and the vesicles that
shuffle various agents to and from the plasma membrane. This system is responsible for
secreting chemicals, moving proteins to appropriate locations (e.g., within the plasma
membrane), as well as modifying the substances that pass through it. All cells contain
multiple mitochondria, which are the powerhouse organelles that are responsible for producing
ATP. As we will see later, the mitochondria also play a major role in initiating programmed
cell death. The last major component is the lysozyme, which digests and removes cellular
waste. For a good review of cellular components, see Alberts et al. (2004), Kandel (2000),
Schwartz and Westbrook (2000), and Brittle and Waters (2000).
The most distinctive characteristic of neurons is the presence of dendrites and an
axon. These structures are responsible for receiving and sending messages between other
neurons. In general, dendrites receive messages, while axons carry messages from the cell
body to other cells. The dendrites contain ion channels that send signals to the cell body of
the neuron; if the signal is strong enough, an action potential will be elicited (Koester &
Siegelbaum, 2000). The action potential travels down the axon to stimulate the release of
chemical messengers from the axon terminal into the synapse. This directionality of signaling
within the neuron gives it a polarity unlike that of any somatic cell. That is not to say that all
somatic cells lack a polarity. The epithelial cells within our gut have distinct differences
between the apical (facing outside the organism) and basal (facing inside the organism)
membranes (see Alberts et al., 2004).
Programmed Cell Death 12
Another quality that sets neurons apart from somatic cells is their supposed
immortality. This is now being debated, and the no-new-neurons dogma may soon be old
news. There is undeniable evidence that neurogenesis takes place during adulthood in several
different brain structures (Luzzati, Marchis, Fasolo, & Peretto, 2006; Hoehn, Palmer, &
Steinberg, 2005; Kokoeva, Yin, & Flier, 2005; for reviews see Zhang, Zhang, & Chopp, 2005;
Gould & Gross, 2002). However, the mechanisms and functions of this neurogenesis are not
as clear as that of somatic cell renewal. Stem cells replace somatic cells as they become
unable to reproduce and eventually die. For an overview of this process, see Alberts et al.
(2004).
But what does any of this have to do with cell death? The function of a cell is
important because it defines its role while alive, but it also indicates how readily an organism
will want to kill it. For example, the cells lining the stomach are in a very harsh environment,
and as such they are replaced every 5 days (Rubin, Saunders, & Kearney, 2006). Neurons
reside in a very controlled environment and make very complex and specific connections that
are not easily replaced, therefore neurons are not replaced as readily.
As we can see, the lifespan is different between neuronal and somatic cells. But
what about how they die? What similarities exist in how the two die, and what makes neurons
live so long? To answer these questions, we must first find out the basic mechanisms of cell
death. First, cell death in somatic cells will be described, then neuronal cell death. For each
cell, the main topics will be how the cells are kept alive and how they die. There will also be
Programmed Cell Death 13
a brief description of what happens when cell death becomes uncontrolled in somatic cells.
Not every method of cell death can be covered, as there are many ways for cells to die; only
the molecular bases of apoptosis will be discussed.
Molecular Basis of Apoptosis
There are many different molecules involved in the control of a cell's life cycle.
Mitogens stimulate cell division, growth factors stimulate cell growth, and survival factors are
involved in keeping the cell from undergoing apoptosis. There are also many signals, derived
both endogenously and exogenously, that induce cell death. Control of cell death is a
complicated interplay between positive and negative signals.
What Keeps a “Normal” Cell Alive?
In order for a cell to live, it must be supplied all of the monomeric components
used to build the necessary complex molecules. For example, a cell building proteins must
contain all of the amino acids that compose the protein. Cellular division is preceded by the
complete duplication of the cellular genome, which requires an abundance of DNA monomers,
deoxyribonucleotides. Carrying out any constructive process requires energy. The form of
energy used by the cells is ATP, a molecule with high-energy phosphate bonds, which is
synthesized by the breakdown of glucose (for review, see Alberts et al., 2004). Without the
energy supplied by ATP, the cell would be unable to create the highly-ordered molecules
necessary to keep it alive. Containing the energy stores and building blocks necessary to
synthesize the life-sustaining molecules is critical for a cell to live.
Programmed Cell Death 14
Cells also receive signals from growth factors, which elicit a number of changes in
cellular physiology that ultimately regulate cell death, differentiation, and, as the name
implies, growth (Alberts et al., 2004; Cook & Urrutia, 2000). Transforming growth factor-β
(TGF-β) directs a diverse array of physiological changes that are cell and environment
specific. TGF-β signaling in certain pancreatic cells reduces growth, while this signaling
may increase proliferation in others. The variety of actions with which TGF-β is responsible
is due to the different downstream signaling proteins it activates or inhibits.
Although there are many different proteins involved in each TGF-β signaling
pathway, there is one common class of proteins – the Smads (Cook & Urrutia, 2000). Smads
come in three varieties: receptor-regulated (R-Smad), co-Smads, and anti-Smads. R-Smads
associate with the TGF-β receptor and are phosphorylated upon binding of TGF-β. The
phosphorylation allows R-Smad to move to the cytoplasm and interact with co-Smads, forming
a complex that regulates gene expression, thus producing cellular changes. Anti-Smads
antagonize R-Smad functioning by disrupting its association with the TGF-β receptor,
interfering with its phosphorylation, or blocking its interaction with co-Smads. The activity of
the Smads accounts for both the specific (e.g., inducing cell death or proliferation) and
variable (e.g., cell type 1 is not affected the same as cell type 2) activity of TGF-β.
Cells also receive death signals that must be removed or concealed in order to
continue to live. The survival factors indirectly create the absence of death signals by
antagonizing their proapoptotic effects. An example of this is the effect of the protein Nur77
Programmed Cell Death 15
on tumor necrosis factor (TNF) signaling. Activation of the TNF receptor initiates a signaling
cascade that ultimately leads to activation of proapoptotic proteins (Arch, Gedrich, &
Thompson, 1998). The apoptotic activity of TNF signaling is negatively regulated by Nur77
(Suzuki et al., 2003). Therefore, Nur77 activity masks TNF, which is equivalent to the
absence of TNF.
How Do Cells Kill Themselves?
The apoptotic machinery exists in all cells, waiting to be activated. The killer
proteins that carry out apoptosis are called caspases. There are two main types of caspases,
the initiator caspases and effector caspases, and both of these exist within the cell in an
inactive zymogen form that must be cleaved in order to function properly (Chang & Yang,
2000). Upon activation, the initiator caspases cleave and activate the effector caspases, as
well as other inactive initiator caspases, creating a positive feedback loop that amplifies the
death signal (Liu, Chang, & Yang, 2004). Effector caspases then act on many other proteins
to produce cell death, including components of the cytoskeleton, RNA splicing factors, and
proteins involved in DNA processing (Thornberry & Lazebnik, 1998). The outcome of
caspase activity is manifested in the cell as morphological and biochemical changes, including
membrane blebbing, as well as condensation and cleavage of the genome (Bouillet & Strasser,
2002).
Another important protein that decides whether or not apoptosis should be induced
is p53, which preserves the genomic integrity of a cell. In the presence of cellular insults, p53
Programmed Cell Death 16
halts cell cycle progression, and if the problem is not alleviated the cell will undergo
apoptosis. An example of cellular insults would be mutated DNA, which leads to p53
activation and eventually cell death if the cell remain mutated. Because of this function, p53
is an essential tumor suppressor. In healthy cells, p53 is normally unstable and transcribed at
very low levels; activation of p53 overcomes these two barriers (Haupt, Berger, Goldberg, &
Haupt, 2003). Through interaction with other cofactors, p53 can induce the expression of
genes involved in the intrinsic apoptotic pathway (e.g., Bax), as well as the extrinsic pathway
(e.g., Fas), acting as a control switch for both types of apoptosis.
Phagocytic cells recognize changes in cells undergoing apoptosis and engulf the
cell to phagocytize, or digest, the dying cells (Fadok et al., 1998). This is a necessary last
step in the apoptotic pathway. The phagocytic response does not, however, produce any
inflammation because apoptotic cells both inhibit the production of inflammatory agents, as
well as produce active anti-inflammatory compounds (Fadok et al., 1998). The lack of an
inflammatory response makes apoptosis a clean way for cells to die, as it does not affect any
surrounding cells. This is advantageous to the organism – imagine if your stomach was
constantly inflamed.
The different signals for cell death can activate different pathways of programmed
cell death. The two main pathways, the intrinsic and extrinsic pathways, differ in their locus
of initiation. The extrinsic pathway is activated by death factors binding to cell surface
Programmed Cell Death 17
“death receptors,” while the intrinsic pathway usually involves an increase in mitochondrial
membrane permeability, which is highly regulated by the Bcl-2 family proteins (Bouillet &
Strasser, 2002).
Intrinsic pathway: Bcl-2 protein-mediated apoptosis. Whether or not a cell
undergoes apoptosis is determined largely by a complex system of interactions between the
Bcl-2 family proteins. The Bcl-2 proteins can be either antiapoptotic (Bcl-2, Bcl-xL, Bcl-w,
Mcl-1, and A1) or proapoptotic, and within the proapoptotic group, they can be directly (Bax
and Bak) or indirectly proapoptotic (the BH3-only proteins Bad, Bik, Blk, Bid, Bim, Bmf,
Noxa, and Puma) (Bouillet & Strasser, 2002). Largely through interactions of the various
BH3-only proteins, the apoptotic fate of cells are modulated by deactivating the antiapoptotic
Bcl-2 proteins and allowing the proapoptotic Bcl-2 proteins to permeabilize the mitochondrial
membrane, causing the release of cytotoxic agents, mainly cytochrome c (Tikhomirov &
Carpenter, 2005). Cytochrome c then interacts with the protein Apaf-1 to activate initiator
caspases (Bouillet & Strasser, 2002). Cell death is carried out as described above.
Programmed Cell Death 18
Once caspases are activated, a
positive feedback loop ensures that their
activity will continue until cell death is
complete. For that reason, caspase activity is
tightly controlled by upstream proteins in the
Bcl-2 family. This is a very important family
of proteins that are categorized by the
presence of at least of the four different BH
(Bcl-2 Homology) domains (Willis, Day,
Hinds, & Huang, 2003). With the exception
Figure 1: Presence of BH (Bcl-2 homology)
domains in the different Bcl-2 proteins.
(from Opferman & Korsmeyer, 2003.)
of the BH3-only proteins, all Bcl-2 proteins contain three or four of the BH domains; the
BH3-only proteins contain only the BH3 domain (see Fig. 1). It is the BH3 domain that is
used to interact with other Bcl-2 proteins within a groove formed by the BH1, BH2, and BH3
domains of the other Bcl-2 proteins
(Willis et al., 2003). As we can see in
Figure 2, the weakly conserved BH3
domain is not big enough to be an
actual domain. It is merely a
collection of a few amino acids
Figure 2: The BH3 domain consists of only a few
poorly conserved amino acids. (from Bouillet &
Strasser, 2002).
Programmed Cell Death 19
responsible for the protein-protein interactions between Bcl-2 proteins.
Further evidence supports the BH3 domain as the domain through which the Bcl-2
proteins interact with each other. First, some BH3-only proteins are capable of activating
Bax (Fletcher & Huang, 2006). It has been shown that it is the BH3 domain of these BH3only proteins that interacts with Bax and ultimately induces the release of cytochrome c
(Opferman & Korsmeyer, 2003). The last piece of evidence that the BH3 domain is the
sequence through which Bcl-2 proteins interact is the fact that some BH3-only proteins,
termed “sensitizers,” indirectly promote apoptosis through interactions with antiapoptotic
Bcl-2 proteins that inhibit their protective function (Fletcher & Huang, 2006; Kelekar et al.,
1997).
The antiapoptotic Bcl-2 proteins prevent oligomerization and permeabilization of
the mitochondrial membrane by the proapoptotic Bax or Bak. The proapoptotic Bcl-2
proteins permeabilize the mitochondrial membrane in one of two ways: by modification of preexisting channel proteins (Marzo et al., 1998), or by forming new pores (Nechushtan, Smith,
Lamensdorf, Yoon, & Youle, 2001). There is still a great deal of mystery in how these
proteins act on the mitochondria, as well as how they are kept from doing so. Adding to this
mystery is the debate as to how exactly the antiapoptotic Bcl-2 proteins inhibit apoptosis.
One model involves direct contacts between the anti- and proapoptotic Bcl-2 proteins on the
mitochondrial membrane that are disrupted by BH3-only proteins, while a second model
proposes contacts between the BH3-only proteins and the antiapoptotic proteins at both the
Programmed Cell Death 20
mitochondria and endoplasmic reticulum (Thomenius & Distelhorst, 2003). In the second
model, the antiapoptotic Bcl-2 proteins inhibit apoptosis indirectly by antagonizing BH3-only
protein activation of the proapoptotic Bcl-2 proteins. This model is supported by the fact
that the antiapoptotic Bcl-2 proteins do not appear to directly interact with Bax or Bak
(Nechushtan et al., 2001). The fact that most BH3-only proteins are sensitizers lends more
support to the first model, though. Whichever model is correct, the antiapoptotic Bcl-2
proteins antagonize apoptotic activity most likely by associating with the mitochondrial
membrane and preventing Bax transposition and oligomerization (Kowaltowski, Cosso,
Campos, & Fiskum, 2002). This protective ability is then disrupted by the actions of the
BH3-only proteins (Kelekaret al., 1997). There is still much to be discovered, but what is
clear is that the antiapoptotic Bcl-2 proteins either directly or indirectly inhibit Bax activity.
There are many more BH3-only proteins than there are effector proteins that
actually prevent or instigate apoptosis. The abundance of modulatory proteins allows the cell
to detect a wide array of apoptotic stimuli, while maintaining a relatively low number of
proteins that actively control the cell's fate. Indeed, a number of experiments have shown the
different BH3-only proteins to respond to specific signals, including cytokine withdrawal,
cellular detachment from matrix, or radiation (Bouillet & Strasser, 2002). The BH3-only
proteins act as cell death signal translators to the effector Bcl-2 proteins, allowing these
proteins to understand the different stimuli as one message: binding of the BH3 domain at the
hydrophobic pocket.
Programmed Cell Death 21
After proapoptotic Bcl-2 proteins are activated and have permeabilized the
mitochondrial membrane, the apoptotic
cytochrome c and Smac enter the cytosol to
activate initiator caspases. Cytochrome c
then interacts with Apaf-1, which then plays
a direct role in caspase activation by
sequestering the inactive initiator caspase
zymogens, forming a structure called the
apoptosome, and acting as an allosteric
activator (Shiozaki, Chai, & Shi, 2002; Shi,
2004). By bringing the inactive caspases
close to one another, a proximity-controlled
mechanism of caspase activation occurs.
Figure 3: Summary of the major molecular
pathways in apoptosis. (from Opferman &
Korsmeyer, 2003.)
There are two proposed mechanisms for this
proximal activation: dimerization, or autolytic cleavage (Shi, 2004; Boatright et al., 2003).
Activated initiator caspases then activate the effector caspases through a series of proteolytic
events (Forsyth, Lemongello, LaCount, Friesen, & Fischer, 2004).
Smac plays a more indirect role in caspase activation. The Inhibitor of Apoptosis
(IAP) family proteins suppress both initiator and effector caspase through direct interactions
(Shi, 2004). The IAPs protect the cell from accidental caspase activation, as this would result
Programmed Cell Death 22
in a deadly positive feedback loop. Smac relieves the IAP inhibition of caspase activity
through direct interactions (Shi, 2004). The combination of cytochrome c and Smac is a
deadly cocktail that results in complete caspase activation.
Now that we have covered the intrinsic pathway of apoptosis, it is time to move on
to the extrinsic pathway. As Figure 3 shows, both of these pathways involve caspase-3
activation, which will ultimately produce apoptosis. Although this diagram shows the extrinsic
pathway (marked Type I) as being less complicated than the intrinsic (Type II) pathway, there
is still much to be discussed.
Extrinsic pathway: Fas receptor-mediated apoptosis. The extrinsic pathway is
activated by the binding of extrinsically-derived factors at surface receptors. This binding at
surface receptors is the critical difference, as the intrinsic pathway is activated by external
signals that do not bind to any surface receptor (e.g., γ-radiation). Ligation at tumor
necrosis factor receptors (TNF-R) induces their oligomerization and is responsible for the
induction of the extrinsic pathway of apoptosis (Bouillet & Strasser, 2002). These receptors
are responsible for eliciting many different responses within the cell, and the complicated
system of protein interactions that follows receptor activation makes it difficult to distinguish
a clear death pathway.
Programmed Cell Death 23
TRAFs (TNF receptor
association factors) are proteins that
interact with the TNF-R, relaying the
death message to other downstream
targets to initiate the apoptotic cascade
(Arch, Gedrich, & Thompson, 1998).
The expression of different TRAFs,
which perform distinct functions, is
specific for each cell type, explaining one
way in which a toxic agent can kill be
harmful to one part of the body, but not
another. However, because there are
Figure 4: The TRAF-mediated portion of the
extrinsic apoptotic pathway. This shows the
oligomerization of TNF-R upon ligation, and
subsequent activation of the TRAFs. (from Arch et
al., 1998.)
many different TRAFs that perform different
functions, this group of proteins is involved in
both cell survival and cell death pathways
(see Fig. 4); we will focus on the apoptotic
aspect of TRAF signaling.
Ligand-bound TNF-R
oligomerizes, causing the recruitment of
Figure 5: Fas signaling links the intrinsic
and extrinsic apoptotic pathways. (from
Pinkoski et al., 2000.)
Programmed Cell Death 24
TRAFs, or their activation via TNF-R-associated death domain proteins (Arch et al., 1998).
Activated TRAFs then form large signaling complexes through protein-protein interactions
with different kinases that initiate a phosphorylation cascade, ultimately leading to c-Jun
amino(N)-terminal kinase (JNK) activation. JNK activity is responsible for eliciting cell death,
as made evident by knockout studies (Yang et al., 1997; Rincon et al., 1998). The apoptotic
activity of JNK is brought about through its activation of the transcription factor c-Jun, as
well as several other downstream effectors (Behrens et al., 2001). Describing all of the
different substrates on which JNK acts and their cellular functions would be a suitable topic
for another thesis; enumerating these is hard enough, as there are many contradictory cell
fates JNK activity can elicit (e.g., cell growth/division or cell death).
The division between
the intrinsic and extrinsic
pathways is not absolute, as the
extrinsic pathway has certain
Bcl-2-dependent components in
its various mechanisms of
caspase activation. Binding at
Fas, a member of the TNF-R
family, activates FADD (fasFigure 6: Diagram of the many signaling pathways
involved in regulating the cell cycle. (from Hanahan &
Weinberg, 2000.)
Programmed Cell Death 25
activated death domain protein), which activates caspase-8 (Pinkoski, Brunner, Green, & Lin,
2000). Activated caspase-8 can then induce apoptosis via a mitochondrial-dependent, or
independent manner. As we can see in Figure 5, the extrinsic and intrinsic pathways are not
totally separate. Although the nomenclature implies two different pathways, they are actually
two different ways of inducing apoptosis. No matter what the classification of apoptosis,
intrinsic or extrinsic, the end product is always caspase activation.
What Happens When Cells Fail to Die?
The bcl-2 gene was discovered because it was overly expressed in cancers; Bcl-2
stands for B-cell lymphoma-2 (Reed, 1994). Therefore, the misregulation of the Bcl-2
proteins, and thus apoptosis, can lead to cancers (specifically B-cell lymphoma). Changes in
the functioning of the Bcl-2 family proteins are not the only ways to produce a cancer. There
are many different changes in cellular functioning that must occur for a cancer to form: the
ability to provide endogenous growth signals, the inability to detect signals that inhibit
growth, the loss of apoptosis, and infinite replicative potential (Hanahan & Weinberg, 2000).
As Figure 6 shows, there are many proteins involved in controlling the cell cycle,
and as such, there are many opportunities for this to go wrong. In order to escape cell cycle
control and become cancerous, the cell must become mutated such that the positive
regulators of cell cycle control (e.g., growth factor receptors) become constitutively active,
while negative regulators of the cell cycle must become inactive (e.g., TNF-R) (Hanahan &
Weinberg, 2000).
Programmed Cell Death 26
Some cell types are more susceptible to the signals that initiate cell death. The
degree to which a certain cell type dies is determined by the ease of its replacement, and how
harsh the environment is in which it lives. Stomach cells are easily replaced and live in a
harsh, acidic environment, therefore they are replaced often. Neurons are not easily replaced
due to their complex, vital connections, and as we will see, they reside in a highly controlled
environment.
Neuronal Specific Cell Death
The life cycle of a neuron involves induction, proliferation, differentiation,
migration, and formation of synaptic connections, after which the neuron will begin to take on
its function (Sastry & Rao, 2000). Neurons spend very little time in the proliferative portion
of their life cycle (before differentiation) as they are useless to the organism until they have
assumed their proper positions and patterns of innervation. Ironically, the most important
part of a neuron's life cycle, functioning physiologically, actually removes the neuron from the
life cycle as it is no longer able to proliferate. Because of the importance of neurons and their
inability to undergo mitosis, these cells are protected to ensure that they live as long as
possible.
What Keeps Neurons Alive?
The vital requirements for neural cells are no different than that of somatic cells.
Neurons still require energy and monomeric components to build all of the necessary
molecules for life. Growth factors are also extremely important in determining the fate of
Programmed Cell Death 27
neuronal cells. TGF-β signaling is responsible for differentiation of developing neuronal
cells, as well as the timing of cell death (Chai, Ito, & Han, 2003). Somatic and neural cells
have the same requirements for life. However, because neurons have much longer expected
lifetimes, there must be some difference in their regulation of life and death.
Like somatic cells, neurons have survival factors called neurotrophic factors that
promote the survival of neurons. The neurotrophic factors are very abundant and well
documented promoters of neuron longevity. The neurotrophins brain-derived neurotrophic
factor (BDNF), nerve growth factor (NGF) and neurotrophins (NT) 3/4/5 are specific
neurotrophic factors for developing neurons (Sastry & Rao, 2000). The different neurotrophic
factors show distinct cell specificities, but all have a similar function – neuron survival
(Arakawa, Sendtner, & Thoenen, 1990). Conversely, the lack of neurotrophic factors can
cause cells to die. This explains why the excess neurons produced prenatally are killed off.
The target cells innervated by neurons secrete neurotrophic factors, but not enough to keep
all neurons alive (Jessell & Sanes, 2000). Only proximal, active neurons will be kept alive, as
only neurons close to the target will have access to neurotrophic factors, and only active
neurons are able to respond properly (Jessell & Sanes, 2000). It is also important to note that
neurons need to have afferent connections as well as efferent connections (Furber,
Oppenhein, & Prevette, 1987). We have all heard the saying “use it or lose it;” this explains
why that is true, as only active cells can properly utilize the neurotrophic factors.
How exactly do neurotrophic factors keep neurons alive? Neurotrophic factors act
Programmed Cell Death 28
as ligands and bind to surface receptors in neurons, activating some intracellular messenger
(Jessell & Sanes, 2000). For example, NGF binds to the trk receptor to activate many
different signaling pathways (e.g., PI-3 kinase, SNT, ras/raf/MAP kinase, PLCγ-1/PKC).
Although there are many different kinases involved, all pathways ultimately activate the
ras/raf/MAP kinase transcriptional pathway, which induces the expression of neuron specific
pro-life genes (Zhu, Hon, Ye, Zhang, 2002). Neurotrophic factors also promote neuron
survival by blocking the activity of apoptotic proteins. Stimulation of neurotrophic receptors
also inhibits caspase activity via antiapoptotic Bcl-2 activation (Jessell & Sanes, 2000). The
finding that increased expression of Bcl-2 compensates for the removal of neurotrophic
growth factor from NGF-dependent neurons in the central nervous system provides further
support that neurotrophic factors promote neuron longevity by disallowing apoptotic
activation (Reed, 1994).
Because the requirements for maintaining proper cellular functioning are the same
in all cells, there are many similarities between neurons and somatic cells in their regulation of
cell death. However, because neurons are not as easily replaced, they have exaggerated
certain aspects in order to ensure a longer lifetime. The glial cells perform many different
functions to help extend the lifetime of neurons. These functions can be reduced to two:
creating an environment free of harmful agents via the blood-brain barrier, or by providing
positive support (e.g., nutrients).
A type of glial cell called astrocytes help form the blood-brain barrier – a
Programmed Cell Death 29
collection of tight junctions between the endothelial cells of the blood vessels within the brain
(Laterra & Goldstein, 2000). While the importance of keeping noxious chemicals from
entering the brain is obvious, the ability of the blood-brain barrier to block the entry of
neurotransmitters circulating in the blood may not be. One of the primary functions of
neurons is to detect signals, and accordingly, they are highly sensitive to their environment.
If the neurotransmitters in the blood were to enter the brain, abnormal signaling, possibly at
cytotoxic levels, could occur. Whereas most water-soluble substances must be actively
transported into the brain, lipid-soluble substances (e.g., O2, CO2, and some drugs) cross the
blood-brain barrier freely (Laterra & Goldstein, 2000). This is very important, as ability to
freely exchanges gases is necessary for life. The selectivity of the blood-brain barrier,
although not perfect, adds control over what enters the brain, without affecting viability.
The glial cells within the brain come in many varieties and serve many functions:
providing the brain with structure, producing myelin, removing cellular debris and secreted
molecules, guiding growing axons, releasing growth factors, and as stated earlier, forming the
blood-brain barrier (Kandel, 2000). Although all of these are important, the release of growth
factors, as we have seen, is responsible for maintaining the life of a neuron. Neurons do not
just receive signals from glial cells; they must communicate with the glial cell in order to elicit
the appropriate response. For example, when neurons are injured, they release fibroblast
growth factor 1, which activates astrocytes and induces the expression of cytoprotective
proteins that promote neuronal survival (Vargas et al., 2005).
Programmed Cell Death 30
How Do Neurons Kill Themselves?
Although neurons have a much more sophisticated and robust system for remaining
alive than somatic cells, they both die in the same way. The Bcl-2 proteins are still very
important regulators of whether or not a cell undergoes apoptosis (Sastry & Rao, 2000). As in
somatic cells, Apaf-1 is an essential signal adaptor in mitochondrial-dependent apoptosis, as
Apaf-1 knockout mice that survived through birth showed abnormal facial growths and
exencephalic brain structures (Yoshida et al., 1998). And most importantly, caspases are still
the executive proteins of apoptosis, as cells without caspase-9 show a reduction in
cytochrome c release, and thus apoptosis (Kuida et al., 1998). The genes encoding caspases
are also expressed in higher amounts in specific cell types when brain injury occurs, giving a
molecular explanation for the cell-specific neurodegeneration associated with certain genetic
diseases (Marciano et al., 2004). As in somatic cells, p53 activity causes cell death. In
neurons, p53 blocks the transcription factor nuclear factor-κB (NF-κB) from expressing the
antiapoptotic Bcl-2 proteins, as well as other apoptotic inhibitors (Culmsee et al., 2003).
As in somatic cells, activity of the kinase JNK is also important in determining the
viability of the cell (Lynn & Wong, 1995). In neurons, an excess of synaptic signaling can
induce cell death. One widely known form of this is the glutamate toxicity seen in the
hippocampal neurons. The neurons of the hippocampus in knockout mice for JNK3, a specific
type of JNK, resisted the toxic effects of excess stimulation of their glutamate receptors
(Yang et al., 1997). As with all other apoptotic pathways observed in somatic cells, JNK
Programmed Cell Death 31
signaling is used to induce neuronal cell death.
The molecular mechanisms for neuronal cell death may be the same as that of
somatic cells, but the reasons for neuronal cell death are debatable. It could be strictly an
issue of providing enough nutrients to the cells and properly shaping the brain structures, or
it could allow the brain to send signals more efficiently. The argument for this side is that
eliminating unnecessary connections increases the speed at which signals can be sent from
one locus to another. This makes sense if we think about the different brain regions as people
and the interconnecting neurons as telephone operators. It would be much quicker to call
someone if you only had to go through 1 or 2 operators rather than 5 or 6. Whatever the
reasons, the fact remains that cell death is an inevitable part of brain development. This
point was made very well by Oppenheim et al. (2001) when they genetically deleted caspase-3
and caspase-9 from several neuron populations. Although cell death was delayed compared to
control groups, the cells killed themselves later by a method not resembling apoptosis to
produce a comparable total amount of cell death between the experimental and control groups
(Oppenheim et al., 2001).
Other Players in Programmed Cell Death
Programmed cell death is a highly controlled process involving interactions
between many different proteins that culminate at the activation or sustained inactivity of
caspases. Although many different apoptotic molecules have been discussed, there are many
more molecular players in programmed cell death, too many, in fact, to thoroughly cover in
Programmed Cell Death 32
this review. The main focus was to outline the most studied and well-known portions of
apoptotic pathways; that does not mean that the smaller branches that deviate from and
connect to this main apoptotic path are not important. Although I will not describe these in
detail, the parenthetically sited sources cover the topics well.
During development, the expression of various Hox genes determines the
morphology of body regions. Hox gene expression inhibits the apoptotic activity of the
Drosophila homologs of Smac – reaper and grim – in migrating neurons (Miguel-Allage &
Thor, 2004). By inhibiting reaper and grim, the IAPs can then inhibit caspase activity and
promote cellular longevity. Hox genes are minor, but important, regulators of caspase
activation.
Calcium ions, Ca2+, appear to play a number of roles in the morphological and
physiological changes associated with cells undergoing apoptosis. Permeabilization of the
mitochondrial membrane allows Ca2+ to leak out into the cytoplasm, raising the internal
concentration of Ca2+. Increased Ca2+ concentration can result in cellular blebbing, chromatin
changes, and induction of Fas-mediated cell death (Trump & Berezesky, 1995). Ca2+ is an
important secondary-messenger in many biological signaling pathways, and as such its role in
cell death is neither surprising, nor indicative of its true biological role. Because of its many
other functions, Ca2+ is not a true death molecule.
To make matters worse for the development of a discrete apoptotic pathway,
recent studies have found connections between the apoptotic and autophagic proteins (Boya
Programmed Cell Death 33
et al., 2005; for review, see Pattingre & Levine, 2006). Although this is inconvenient for
anyone trying to fully understand apoptosis, it is not a surprise. Apoptosis is closely tied to
almost every cellular process that affects the well-being of the cell (i.e., everything a cell
does). The current research suggests that the apoptotic proteins are involved in autophagy,
but unpublished work by Adam Oberstein in Yigong Shi's lab at Princeton University
demonstrates that the interaction between the two systems is a two-way street; autophagic
proteins also regulate apoptosis.
With all of the different proteins and cofactors associated with apoptosis, it may be
hard to understand what the actual death pathway is. The reason for the almost innumerable
apoptotic agents is not just to muddle the pathway; each new protein provides a new level of
controlling cell death. It is vital for an organism, and especially its cells, that cell death be as
programmed as possible. Although apoptosis may seem like a process in which any protein
can potentially be involved, its key components become clear once we study a simplified
system. This is not hard to do, we just need to look to a less complex life form than
mammals. As is the case with most cellular processes, the basic mechanism is the same
between humans and simple organisms, but what differs is the complexity of the control
points. Our stripped down version of apoptosis will be that of Caenorhabditis elegans, the
nematode.
Programmed Cell Death 34
Apoptosis Summary: A Lesson
From Worms
Now that we
understand how mammalian cells
decide to kill themselves, looking
at the apoptotic pathway of C.
elegans will be like child's play.
A common theme in the evolution
Figure 8: Crystal structures comparing an insect effector
caspase (Sf-c1) to different human effector caspases (Hs-c).
(from Forsyth et al., 2004.)
of cellular processes is that as
the organism becomes more
complex, their arsenal of proteins
becomes much larger. If we look at
just the Bcl-2 homologs in C.
elegans, we find that the nematode
Figure 7: Comparison of the apoptotic pathways in
organisms of different complexities. Apoptosis in
Drosophila is an intermediate between that of C.
elegans and mammals. (from Shi, 2004.)
contains two proteins, whereas mammalian cells contain at least twenty-one (see Figure 1).
We have evolved more proteins because we also have many different, highly-specialized cell
types. With less proteins, it is much easier to understand how apoptosis works in C. elegans
(see Figure 7).
The integration of apoptotic signals is highly simplified in C. elegans, as the Bcl-2
family of proteins has been reduced in numbers. The BH3-only and antiapoptotic Bcl-2
Programmed Cell Death 35
family proteins are replaced by EGL-1 and CED-9, respectively (Shi, 2004). CED-4 is
homologous to Apaf-1, and all of the caspases are replaced by CED-3 (Shi, 2004). As Figure
8 shows, homologs (boxes with the same color) do not exist in C. elegans for Smac or the
IAPs, both of which add an additional level of control to carrying out programmed cell death
(Shi, 2004). The reduction in numbers of proteins that perform the same function, as well as
removal of entire groups of proteins, has made it much easier to identify the necessary
components of the apoptotic pathway.
Comparing the apoptotic proteins between species allows us to clarify which ones
are necessary for regulating cell death. Because the homologous, conserved proteins perform
the same function, we would expect their structures to also be conserved. Figure 8 shows the
striking similarities between the caspase of an insect, Spodoptera frugiperda, and different
human caspases. The shape of a molecule determines its function, and shape change is the
method by which protein activation/deactivation is accomplished. The conformational
changes associated with caspase activation are well studied, however they are beyond the
scope of this paper (for review, see Shi, 2004).
Conclusion
We have seen the complex interactions that control the life and death of our cells,
as well as the bare-bones version present in the primitive nematode. While protein signaling
cascades are interesting, there are more useful reasons for studying the regulation of
apoptosis. The most obvious and applicable issue to which research on cell death can be
Programmed Cell Death 36
applied is cancer. Cancer is the absence of cell death, and if we can identify the usual
suspects in the induction of cell death, we will have a better idea of what aberrations to
search for and possibly correct in cancerous cells.
On the other hand, identifying the culprits in eliciting cell death can allow us to
inhibit unwanted cell death (e.g., excessive neurodegeneration). The β-amyloid plaques
observed in patients with Alzheimer's disease have been reported to cause the cell death that
brings about the cognitive disabilities. Morishima et al. (2001) showed evidence that the βamyloid induced cell death was carried out via the extrinsic apoptotic pathway. However,
Alzheimer's disease is poorly understood, and there are many contradictory findings.
Therefore, any finding should be taken as incomplete as best. It is obvious that Alzheimer's
disease induces cell death, but the exact mechanism is not known.
Research on programmed cell death is a widely-applicable and diverse field.
Research topics can be broad, like cancer, or more specific, like the DNA mismatch repair
system. Whatever aspect of apoptosis is studied, it will contribute to a greater understanding
of this important cellular process. The application of apoptotic research has the potential to
improve the quality of many people's lives, and possibly prevent their premature death. Our
understanding of apoptosis will lead to a brighter future.
References
Alberts, B., Bray, D., Hopkin, K., Johnson A., Lewis, J., Raff, M., et al. (2004). Essential cell biology (2nd ed.).
Programmed Cell Death 37
New York: Garland Science.
Arakawa, Y., Sendtner, M., & Thoenen, H. (1990). Survival effect of ciliary neurotrophic factor (CNTF) on chick
embryonic motoneurons in culture: comparison with other neurotrophic factors and cytokines. Journal of
Neuroscience, 10, 3507-3515.
Arch, R.H., Gedrich, R.W., & Thompson, C.B. (1998). Tumor necrosis factor receptor-associated factors
(TRAFs) – a family of adapter proteins that regulates life and death. Genes and Development, 12(18),
2821-2830.
Behrens, A., Sabapathy, K., Graef, I., Leary, M., Crabtree, G.R., & Wagner, E.F. (2001). Jun N-terminal kinase
2 modulates thymocyte apoptosis and T cell activation through c-Jun and nuclear factor of activated T
cell (NF-AT). Proceedings of the National Academy of Sciences of the United States of America, 98 (4),
1769-1774.
Bhatia, M. (2003). Apoptosis versus necrosis in acute pancreatitis. American Journal of Physiology –
Gastrointestinal and Liver Physiology, 286, G189-G196.
Boatright, K.M., Renatus, M., Scott, F.L., Sperandio, S., Shin, H., Pedersen, I.M., et al. (2003). A unified model
for apical caspase activation. Molecular Cell, 11, 529-541.
Bouillet, P. & Strasser, A. (2002). BH3-only proteins – evolutionarily conserved proapoptotic Bcl-2 family
members essential for initiating programmed cell death. Journal of Cell Science, 115(8), 1567-1574.
Boya, P., González-Polo, R.A., Casares, N., Perfettini, J.L., Dessen, P., Larochette, N., et al. (2005). Inhibition
of macroautophagy triggers apoptosis. Molecular and Cellular Biology, 25(3), 1025-1040
Brittle, E.E. & Waters, M.G. (2000). ER-to-golgi traffic – This bud's for you. Science, 289(5478), 403-404.
Bröker, L.E., Kruyt, F.A.E., & Giaccone, G. (2005). Cell death independent of caspases: A review. Clinical
Cancer Research, 11(9), 3155-3162.
Campbell, N.A., & Reece, J.B. (2002). Biology (6th ed.). San Fransisco: Benjamin Cummings.
Chai, Y., Ito, Y., & Han, J. (2003). TGF-β signaling and its functional significance in regulating the fate of
Programmed Cell Death 38
cranial neural crest cells. Critical Reviews in Oral Biology and Medicine, 14 (2), 78-88.
Chang, H.Y., & Yang, X. (2000). Proteases for cell suicide: Functions and regulation of caspases. Microbiology
and Molecular Biology Reviews, 64(4), 821-846.
Cook, T. & Urrutia, R. (2000). TIEG proteins join the Smads as TGF-β-regulated transcription factors that
control pancreatic cell growth. American Journal of Physiological: Gastrointerstinal and Liver
Physiology, 278, G513-G521.
Culmsee, C., Siewe, J., Junker, V., Retiounskaia, M., Schwarz, S., Camandola, S., et al. (2003). Reciprocal
inhibition of p53 and nuclear factor-κB transcriptional activities determines cell survival or death in
neurons. The Journal of Neuroscience, 23(24), 8586-8595.
del Riccio, V., van Tutyl, M., & Post, M. (2004). Apoptosis in lung development and neonatal lung injury.
Pediatric Research, 55, 183-189.
Fadok V.A., Bratton, D.L., Konowal, A., Freed, P.W., Westcott, J.Y., & Henson, P.M. (1998). Macrophages
that have ingested apptotic cells in vitro inhibit proinflammatory cyokine production through
autocrine/paracrine mechansisms involving TGF-β, PGE2, and PAF. The Journal of Clinical
Investigation, 101(4), 890-898.
Fletch, J.I. & Huang, D.C.S. (2006). BH3-only proteins: Orchestrating cell death. Cell Death and
Differentiation, 1-4.
Forsyth, C.M., Lemongello, D., LaCount, D.J., Friesen, P.D., & Fisher, A.J. (2004). Crystal structure of an
invertebrate caspase. The Journal of Biological Chemistry, 279(8), 7001-7008.
Furber, S., Oppenheim, R.W., & Prevette, D. (1987). Naturally-occurring neuron death in the ciliary ganglion of
the chick embryo following removal of preganglionic input: Evidence for the role of afferents in ganglion
cell survival. The Journal of Neuroscience, 7(6), 1816-1832.
Gould, E., & Gross, C.G. (2001). Neurogenesis in adult mammals: Some progress and problems. The Journal of
Neuroscience 22(3), 619-623.
Programmed Cell Death 39
Hanahan, D. & Weinberg, R.A. (2000). The hallmarks of cancer. Cell, 100, 57-70.
Haupt, S., Berger, M., Goldberg, Z., & Haupt, Y. (2003). Apoptosis – the p53 network. Journal of Cell Science,
116(20), 4077-4085.
Hoehn, B.D., Palmer, T.D., & Steinberg, G.K. (2005). Neurogenesis in rats after focal cerebral ischemia is
enhanced by indomethacin. Stroke, 36, 2718-2724.
Jessell, T.M. & Sanes J.R. (2000). The generation and survival of nerve cells. In E.R. Kandel, J.H. Schwartz, &
T.M. Jessell (Eds.), Principles of Neural Science (4th ed.) (1041-1062). New York: McGraw-Hill.
Kandel, E.R. (2000). Nerve cells and behavior. In E.R. Kandel, J.H. Schwartz, & T.M. Jessell (Eds.), Principles of
Neural Science (4th ed.) (19-35). New York: McGraw-Hill.
Kelekar, A., Chang, B.S., Harlan, J.E., Fesik, S.W., & Thompson, C.B. (1997). Bad is a BH3 domain-containing
protein that forms an inactivating dimer with Bcl-xL. Molecular and cellular Biology, 17 (2), 7040-7046.
Koester, J. & Siegelbaum, S.A. (2000). Propagated signaling: The action potential. In E.R. Kandel, J.H.
Schwartz, & T.M. Jessell (Eds.), Principles of Neural Science (4th ed.) (150-170). New York: McGrawHill.
Kokoeva, M.V., Yin, H., & Flier, J.S. (2005). Neurogenesis in the hypothalamus of adult mice: Potential role in
energy balance. Science, 310(5748), 679-683.
Kowaltowski, A.J., Cosso, R.G., Campos, C.B., & Fiskum, G. (2002). Effect of Bcl-2 overexpression on
mitochondrial structure and function. The Journal of Biological Chemistry, 277(45), 42802-42807.
Kresch, M.J., Christian, C., Wu, F., & Hussain, N. (1998). Ontogeny of apoptosis during lung development.
Pediatric Research, 43, 426-431.
Kuida, K., Haydar, T.F., Kuan, C.Y., Gu, Y., Taya, C., Karasuyama, H., et al. (1998). Reduced apoptosis and
cytochrom c-mediated caspase activation in mice lacking caspase 9. Cell, 94, 325-337.
Laterra, J. & goldstein, G.W. (2000). Ventriular organization of cerebrospinal fluid: Blood-brain barrier, brain
edema, and hydrocephalus. In E.R. Kandel, J.H. Schwartz, & T.M. Jessell (Eds.), Principles of Neural
Programmed Cell Death 40
Science (4th ed.) (1288-1301). New York: McGraw-Hill.
Liu, H, Chang, D.W., & Yang, X. (2004). Interdimer processing and linearity of procaspase-3 activation. The
Journal of Biological Chemistry, 280(12), 11578-11582.
Luzzati, F., Marchis, S.D., Fasolo, A., & Peretto, P. (2006) Neurogenesis in the caudate nucleus of the adult
rabbit. The Journal of Neuroscience, 26(2), 609-621.
Lynn, W.S., & Wong, P.K.Y. (1995). Neuroimmunodegeneration: Do neurons and T cells use common pathways
for cell death? The Federation of American Societies for Experimental Biology Journal, 9 , 1147-1156.
Marciano, P.G., Brettschneider, J., Manduchi, E., Davis, J.E., Eastman, S., Raghupathi, R., et al. (2004).
Neuron-specific mRNA complexity responses during hippocampal apoptosis after traumatic brain injury.
The Journal of Neuroscience, 24(12), 2866-2876.
Marzo, I., Brenner, C., Zamzami, N., Jürgensmeier, J.M., Susin, S.A., Veira, H.L.A., et al. (1998). Bax and
adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science, 281,
2027-2031
Miguel-Allaga, I. & Thor, S. (2004). Segment-specific prevention of pioneer neuron apoptosis by cellautonomous, postmitotic Hox gene activity. Development, 131(24), 6093-6105.
Morishima, Y., Gotch, Y., Zieg, J., Barrett, T., Takano, H., Flavell, R., et al. (2001). β-amyloid induces
neuronal apoptosis via a mechanism that involves the c-Jun N-terminal kinase pathway and the induction of
Fas ligand. The Journal of Neuroscience, 21(19), 7551-7560.
Nechushtan, A., Smith, C.L., Lamensdorf, I., Yoon, S.H., & Youle, R.J. (2001). Bax and Bak coalesce into novel
mitochondrial-associated clusters during apoptosis. The Journal of Cell Biology, 153(6), 1265-1276.
Opferman, J.T., & Korsmeyer, S.J. (2003). Apoptosis in development and maintenance of the immune system.
Nature Immunology, 4(5), 410-415.
Oppenheim, R.W., Flavell, R.A., Vinsant, S., Prevette, D., Kuan, C.Y., & Rakic, P. (2001). Programmed cell
death of developing mammalian neurons after genetic deletion of caspases. The Journal of Neuroscience,
Programmed Cell Death 41
21(13), 4752-4760.
Pattingre, S. & Levine, B. (2006). Bcl-2 inhibition of autophagy: A new route to cancer? Cancer Research,
66(6), 2885-2888.
Pinkoski, M.J., Brunner, T., Green, D.R., & Lin, T. (2000). Fas and Fas ligand in gut and liver. The American
Journal of Physiology: Gastrointestinal and Liver Physiology, 278 , G354-G366.
Qu, Z., Yu, J., Bhagat, G., Furuya, N., Hibshoosh, H., Troxel, A., et al. (2003). Promotion of tumorigenesis by
heterozygous disruption of the beclin 1 autophagy gene. The Journal of Clinical Investigation, 112(12),
1809-1820.
Rabinowicz, T., de Courten-Myers, G.M., Petetot, J.M., Xi, G., & de los Reyes, E. (1996). Human cortex
development: estimates of neuronal numbers indicate major loss late during gestation. Journal of
Neuropathology and Experimental Neurology, 55(6), 320-328.
Reed, J.C. (1994). Bcl-2 and the regulation of programmed cell death. The Journal of Cell Biology, 124(1 & 2),
1-6.
Rincon, M., Whitmarsh, A., Yang, D.D., Weiss, L., Derijard, B., Jayaraj, P., et al. (1998). The JNK pathway
regulates the in vivo deletion of immature CD4+CD8+ thymocytes. The Journal of Experimental
Medicine, 188(10), 1817-1830.
Rubin, C.E., Saunders, D.R., & Kearney, D.J. (2006). Stomach. In The Gut Course: On-line Syllabus (III).
Retrieved October 14, 2006 from http://www.uwgi.org/gut/stomach_01.asp.
Sastry, P.S. & Rao K.S. (2000). Apoptosis and the nervous system. Journal of Neurochemistry, 74(1), 1-20.
Schwartz, J.H., & Westbrook, G.L. (2000). The cytology of neurons. In E.R. Kandel, J.H. Schwartz, & T.M.
Jessell (Eds.), Principles of Neural Science (4th ed.) (67-87). New York: McGraw-Hill.
Shi, Y. (2004). Caspase activation, inhibition, and reactivation: A mechanistic view. Protein Science, 13, 19791987.
Shiozaki, E.N., Chai, J., & Shi, Y. (2002). Oligomerization and activation of caspase-9, induced by Apaf-1
Programmed Cell Death 42
CARD. Proceedings of the National Academy of Sciences of the United States of America, 99 (7), 41974202.
Sugden, P.H. & Clerk, A. (1998). “Stress-responsive” mitogen-activated protein kinases (c-Jun N-terminal
kinases and p38 mitogen activated protein kinases) in the mitocardium. Circulation Research, 83, 345352.
Suzuki, S., Suzuki, N., Mirtsos, C., Horacek, T., Lye, E., Noh, S.K., et al. (2003). Nur77 as a survival factor in
tumor necrosis factor signaling. Proceedings of the National Academy of Sciences of the United States
of America, 100(14), 8276-8280.
Thomenius, M.J. & Distelhorst, C.W. (2003). Bcl-2 on the endoplasmic reticulum: protecting the mitochondria
from a distance. Journal of Cell Science, 116(22), 4493-4499.
Thornberry, N.A. & Lazebnik, Y. (1998). Caspases: enemies within. Science, 281, 1312-1316.
Tikhomirov, O. & Carpenter, G. (2005). Bax activation and translocation to mitochondria mediate EGF-induced
programmed cell death. Journal of Cell Science, 118, 5681-5690.
Trump, B.F., & Berezesky, I.K. (1995). Calcium-mediated cell injury and cell death. The Federation of American
Societies for Experimental Biology Journal, 95, 219-228.
Vargas, M.R., Pehar, M., Cassina, P., Martinez-Palma, L., Thompson, J.A., Beckman, J.S., et al. (2005).
Fibroblast growth factor-1 induces heme oxygenase-1 via nuclear factor erthroid 2-related factor
2(Nrf2) in spinal cord astrocytes. The Journal of Biological Chemistry, 280(27), 25571-25579.
Willis, S., Day, C.L., Hinds, M.G., & Huang, D.C.S. (2003). The Bcl-2 regulated apoptotic pathway. Journal of
Cell Science, 116(20), 4053-4056.
Yang, D.D., Kuan, C.Y., Whitmarsh, A.J., Rinocn, M., Zheng, T.S., Davis, R.J., et al. (1997). Absence of
excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene. Nature, 389, 865870.
Yoshida, H., Kong, Y.Y., Yoshida, R., Elia, A.J., Hakem, A., Hakem, R., et al. (1998). Apaf1 is required for
Programmed Cell Death 43
mitochondrial pathways of apoptosis and brain development. Cell, 94, 739-750.
Yu, L., Alva, A., Su, H., Dutt, P., Freundt, E., Welsh, S., et al. (2004). Regulation of an ATG7-beclin 1
program of autophagic cell death by caspase-8. Science, 304, 1500-1502.
Yue, Z., Jin, S., Yang, C., Levine, A.J., & Heintz, N. (2003). Beclin 1, an autophagy gene essential for early
embryonic development, is a haploinsufficient tumor suppressor. Proceeding of the National Academy of
Sciences, 100(25), 15077-15082.
Zhang, R.L., Zhang, Z.G., & Chopp, M. (2005). Neurogenesis in the adult ischemic brain: Generation, migration,
survival, and restorative therapy. The Neuroscientist, 11, 408-416.
Zhang, Y., Dimtehev, A., Dritschilo, A., & Jung, M. (2001). Ionizing radiation-induced apoptosis in ataxiatelangiesctasia fibroblasts. The Journal of Biological Chemistry, 276(31), 28542-28548.
Zhu, Y., Hon, T., Ye, W., & Zhang, L. (2002). Heme deficiency interferes with the ras-mitogen-activated
protein kinase misnaling pathway and expression of a subset of neuronal genes. Cell Growth and
Differentiation, 13, 431-439.
Zong, W.X., & Thompson, C.B. (2006). Necrotic death as a cell fate. Genes and Development, 20, 1-15.