ScienceWeek

SCIENCEWEEK

ScienceWeek
June 20, 2003
Vol. 7 Number 25A

An Online Digest of Research in the Sciences

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Our biology has made us into creatures who are constantly
recreating our psychic and material environments, and whose
individual lives are the outcomes of an extraordinary
multiplicity of intersecting causal pathways. Thus, it is our
biology that makes us free.
-- Richard Lewontin

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Section 1

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Part A (Symposium) is transmitted by Email on Mondays.
Part B (News & Views) is transmitted by Email on Wednesdays.
Part C (Quark - New Books & Miscellany) is transmitted by Email
on Fridays. All parts carry the Friday date.

The HTML version of this file is available at:
http://www.scienceweek.com/2003/sw030620.htm


Part A - Symposium: Cell Biology: Apoptosis

1. Introduction
2. Caspases and Apoptosis
3. Mitochondria and Apoptosis
4. Apoptosis in Development
5. Apoptosis in the Nervous System
6. Apoptosis and Clinical Medicine

Notices and Subscription Information

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Section 2

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1. INTRODUCTION

As of this writing, the world literature contains more than
61,000 research papers on apoptosis, nearly all of them published
during the past two decades.

"Apoptosis" (programmed cell death) is a rapid and specific
process involving the production of a number of enzymes in the
cell programmed to be destroyed. This programmed destruction is
not always harmful, or always the result of cellular damage of
one sort or another. In humans, for example, the lack of webbing
between fingers and toes is a result of apoptosis of cells of
webbing tissue occurring during embryological development, the
apoptosis in this case being a normal part of the larger
embryological program. In the mature organism, apoptosis is the
usual method of removing damaged cells after these cells are
recognized to be damaged by one mechanism or another. It is known
that normal cells carry an apoptosis receptor on their surfaces,
called CD95, and that when this surface receptor is cross-linked
by its specific ligand, this triggers the sequence of events
known as apoptosis. In the apoptosis sequence, certain
proteolytic enzymes inside the cell are activated, and in
addition a variety of lipids that cause cell dysfunction are
synthesized.

NOTES ON TERMINOLOGY

In the word "apoptosis", both "p"s are often pronounced. The term
derives from the Greek ptosis  "a falling".  The other type of
cell death, "necrosis", is a pathological death caused by
external factors such as toxic injury. The phenomenon of natural
cell death has been noted off and on since the 19th century, but
the term "apoptosis" was apparently first used to describe the
phenomenon in cells in 1972 (J.F. Kerr et al: Brit. J. Cancer
1972 26:239). In clinical medicine, the term "ptosis" or the
suffix "-ptosis" refers to the prolapse (falling down) of a
tissue or organ; the clinical usage of "ptosis" has no relevancy
to the usage of "apoptosis" in cell biology. In clinical
medicine, the "p" in ptosis is silent.

"Mitochondria" are double-membrane enclosed organelles of cells
that are involved with several important biochemical pathways,
including electron transport and oxidative metabolism. Various
types of eukaryotic cells (cells with nuclei) may contain from a
few to several thousand mitochondria in each cell type. The
mitochondria are relatively large cylindrical structures up to 10
microns long and up to 2 microns in diameter, and most biologists
believe mitochondria are cell organelles that may have originated
as separate organisms that became resident in eukaryotic cells.
Mitochondrial DNA is independent of nuclear DNA. It consists of a
circular molecule, 16,569 base pairs long in humans, with a known
nucleotide sequence.

"Proteolytic enzymes", also called "proteases", split proteins
and thereby degrade them. The enzymes catalyze the hydrolysis of
peptide bonds, fragmenting proteins into polypeptide chains, and
fragmenting polypeptide chains into constituent amino acids.
Sometimes proteolytic enzymes and proteases are distinguished,
with the term "proteases" reserved for proteolytic enzymes with
high specificity for peptide bonds between particular amino
acids.

The term "membrane blebbing" refers to the macroscopic blistering
of the surfaces of cells when they die under certain conditions.

The term "endoplasmic reticulum" refers to a complex system of
flattened sacs in all biological cells that have a nucleus
(eukaryotes). The endoplasmic reticulum is the site of many
important syntheses, including the production of new surface
membrane and the intracellular transport of various biochemical
entities.

The "cytochromes" are a system of electron-transfer proteins with
iron- or copper-porphyrin as a prosthetic group. They are found
in both animal and plant cells. Cytochrome-c is found in
mitochondria.

ON APOPTOSIS

Programmed cell death, or apoptosis, is currently one of the
hottest areas of modern biology. It describes the orchestrated
collapse of a cell, staging membrane blebbing, cell shrinkage,
protein fragmentation, chromatin condensation and DNA degradation
followed by rapid engulfment of corpses by neighboring cells.

The excitement ensued when it became clear that apoptosis is an
essential part of life for any multicellular organism and that
the way in which most cells die is conserved from worm to mammal.
Optimum body maintenance means that about 10 billion of our cells
will die on a normal day just to counter the numbers of new cells
that arise through mitosis. During development apoptosis helps to
sculpture the body, shape the organs, and carve out fingers and
toes. Both the nervous system and the immune system arise through
overproduction of cells followed by the death of those that fail
to establish functional synaptic connections or productive
antigen specificities, respectively. Apoptosis is necessary to
purge the body of pathogen-invaded cells, but is also needed to
eliminate activated or auto-aggressive immune cells.

Such massacre has to be tightly regulated as too little or too
much cell death may lead to pathology, including developmental
defects, autoimmune diseases, neurodegeneration or cancer. It is
not surprising then that the hunt is on to understand precisely
which cells die when, why, and how, and to find drugs that
interfere with specific steps along the pathway.

Adapted from: Marie-Therese Heemels: Nature 2000 407:769

APOPTOSIS AND THE STABILITY OF LIFE

Individual biological cells in multicellular organisms face three
choices: to divide (mitosis), to specialize (differentiation), or
to commit suicide (apoptosis), and the balance between these
choices ensures tight regulation of cell numbers within
organisms. As a consequence of continuing ordinary mitosis
without cell death, for example, an 80-year-old person would have
2 tons of bone marrow and lymph nodes and an intestinal tract 16
kilometers long. Since apoptosis is more than 20 times faster
than mitosis, sightings of cells dying from apoptosis are rare.
In contrast to passive cell death (necrosis), which is
characterized by leakage and inflammation, apoptotic cells are
engulfed and degraded by neighboring cells without a trace.
Various morphologies currently regarded as apoptotic have been
observed since the 19th century, but it was not until the 1980s
that apoptosis was recognized as a specific process when R.
Horvitz et al mapped the fate of every cell in the nematode worm
C. elegans, including those cells committed to die. It emerged
that programmed cell death is determined by a handful of genes,
and that these "master switches" have been conserved in evolution
so that they, or rather their equivalent gene families, still
orchestrate apoptosis in mammals. The idea has gradually emerged
that the stability of the body is maintained by signals that
control life and death of single cells. This is a powerful
concept, implying that there are specific survival and death
signals, and corresponding receptors on cell surfaces. Such
social control of life and death are vital in complex
multicellular networks such as the immune system and the nervous
system, where communication between cells is crucial.

Adapted from: Gerry Melino: Nature 2001 412:23

ON PROGRAMMED CELL DEATH

"To be, or not to be: that is the question." While we all are
poised at life-or-death decisions, this existential dichotomy is
exceptionally stark for embryonic cells. Programmed cell death,
called apoptosis, is a normal part of development. In the
nematode worm C. elegans, in which we can count the number of
cells, exactly 131 cells die according to the normal
developmental pattern. All the cells of this nematode are
"programmed" to die unless they are actively told not to undergo
apoptosis. In humans, as many as 10^(11) cells die in each adult
each day and are replaced by other cells. (Indeed, the mass of
cells we lose each year through normal cell death is close to our
entire body weight!) Within the uterus, during our fetal
development, we were constantly making and destroying cells, and
we generated about three times as many neurons as we eventually
ended up with when we were born.

Apoptosis is necessary not only for the proper spacing and
orientation of neurons, but also for generating the middle ear
space, the vaginal opening, and the spaces between our fingers
and toes. Apoptosis prunes away unneeded structures, controls the
number of cells in particular tissues, and sculpts complex
organs.

Different tissues use different signals for apoptosis. One of the
signals often used in vertebrates is bone morphogenetic protein 4
(BMP4). Some tissues, such as connective tissue, respond to BMP4
by differentiating into bone. Others, such as the frog gastrula
ectoderm, respond to BMP4 by differentiating into skin. Still
others, such as neural crest cells and tooth primordia, respond
by degrading their DNA and dying. In the developing tooth, for
instance, numerous growth and differentiation factors are
secreted by the enamel knot. After the cusp has grown, the enamel
knot synthesizes BMP4 and shuts itself down by apoptosis.

In other tissues, the cells are "programmed" to die, and they
will remain alive only if some growth or differentiation factor
is present to "rescue" them. This happens during the development
of mammalian red blood cells. The red blood cell precursors in
the mouse liver need the hormone erythropoietin in order to
survive. If they do not receive it, they undergo apoptosis. The
erythropoietin receptor works through the JAK-STAT pathway,
activating the Stat5 transcription factor. In this way, the
amount of erythropoietin present can determine how many red blood
cells enter the circulation.

Adapted from: Scott F. Gilbert: Developmental Biology. 6th
Edition. Sinauer 2000, p.165.

ON CELL DEATH AND AGING

"Cell death is not restricted to embryonic development. As
organisms grow older, cells begin to deteriorate and die. The
possibility that the process of cell aging and death is under
genetic control was first suggested in 1961 when Leonard Hayflick
reported that normal human fibroblasts have a built-in limit to
the number of times they can proliferate. His experiments
revealed that fibroblasts taken from an embryo and grown in
culture divide about 50 times before they deteriorate and die. In
contrast, fibroblasts taken from adults multiply only 15-30 times
before dying. And fibroblasts isolated from young children
suffering from Werner's syndrome, a rare disease that causes
youngsters to age prematurely, divide only 2-10 times in culture.
Further evidence for a relationship between aging and a cell's
proliferative capacity came with the discovery that the number of
times a cell can divide in culture is related to the life span of
the organism. Thus cells of the Galapagos tortoise, whose maximum
life span is about 175 years, divide more than a 100 times in
culture before dying, whereas cells obtained from mice, whose
life expectancy is only a few years, divide fewer than 30 times."

L.J. Kleinsmith and V.M. Kish: Principles of Cell and Molecular
Biology. 2nd Edition. HarperCollins 1995, p.709.

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2. CASPASES AND APOPTOSIS

THE PROTEASE CASPASE-2 IN APOPTOSIS

Science 2002 297:1290

The following points are made by S. Kumar and D.L. Vaux:

1) Activation of proteolytic enzymes called "caspases" is a key
step in the apoptotic program. Caspases exist in latent forms in
almost all animal cells and become activated in response to
apoptotic signals such as those induced by cell stress (for
example, DNA damage and withdrawal of trophic support). The first
caspases to become activated are so-called "initiator caspases".
These caspases have long amino-terminal prodomains containing
specific protein-protein interaction motifs. Through these
domains the caspases interact with adaptor proteins that recruit
them to specific "death complexes" (large multiprotein complexes
that mediate caspase activation). In mammals, these death
complexes include the Apaf-1/caspase-9 apoptosome and the
FADD/caspase-8 death-inducing signaling complex (DISC).

2) Once the initiator caspases are activated, they process and
activate downstream effector caspases, such as caspases 3, 6, and
7 (1). The apoptosome and DISC are thought to account for most
caspase-dependent apoptosis. The upstream signaling pathways
leading to the assembly of these death complexes are often called
the mitochondrial (intrinsic) and death receptor (extrinsic)
pathways of apoptosis.

3) During stress-induced apoptosis, mitochondria release their
cytochrome c, which binds to Apaf-1 and promotes apoptosome
formation and caspase-9 activation (1). Thus, the most widely
held view is that caspase-9 is the initiator caspase in this
pathway and that mitochondrial release of cytochrome c is
essential for activation of this caspase (1,2). However, this
view has been challenged [see (2)] and is now under challenge
again from several reports (3-5). For example, in response to
cell stress, activation of a neglected caspase, caspase-2, is
required before mitochondrial permeabilization and apoptosis can
take place (3).

4) Caspase-2 (Nedd2/Ich-1) was the first mammalian apoptotic
caspase to be identified. It closely resembles caspase-9 and CED-
3 in the worm, and like them bears a caspase recruitment domain
(CARD). Numerous early studies implicated caspase-2 in cell death
pathways that could be rescued by Bcl-2. Caspase-2 is
ubiquitously expressed, its activation occurs early in apoptosis,
and antisense studies suggest that reducing the amount of
caspase-2 lessens cell death in response to factor deprivation of
cultured cells and sympathetic neurons.

References (abridged):

1. N. A. Thornberry, Y. Lazebnik, Science 281, 1312 (1998)

2. E. Finkel, Science 292, 624 (2001)

3. P. Lassus, X. Opitz-Araya, Y. Lazebnik, Science 297, 1352
(2002)

4. Y. Guo, S. M. Srinivasula, A. Druilhe, T. Fernandes-Alnemri,
E. S. Alnemri, J. Biol Chem. 277, 13430 (2002)

5. J. D. Robertson, M. Enoksson, M. Suomela, B. Zhivotovsky, S.
Orrenius, J. Biol. Chem. 277, 29803 (2002)

Related Material:

ON THE BIOCHEMISTRY OF CASPASES

Chem. Rev. 2002 102:4489

The following points are made by J-B. Denault and G.S. Salvesen:

1) Cell death is obligatory for harmonious cell life in animals,
from the cavitation of the early embryo to the removal of
infected or cancerous cells in the adult. Although there is more
than one way for any given cell to die, it is generally the
apoptotic mechanism that is used by the organism because it is a
tidy, organized, and inflammation-free process. At the center of
this death process is a family of proteases named caspases (the
"c" is intended to reflect a cysteine protease mechanism, and
"aspase" refers to their ability to cleave after aspartic acid,
the most distinctive catalytic feature of this protease family).
Members of the family are found in worms to flies to humans and
can be traced back to distantly related proteases from
Porphyromonas gingivalis.

2) In humans, caspases are ubiquitously expressed cytosolic
proteases synthesized as latent zymogens awaiting an appropriate
activation stimulus. Whereas seven members (caspases 2, 3, and 6-
10) are usually considered to be part of the apoptotic machinery,
three others (caspases 1, 4, and 5) are employed by another
innate defense mechanism, the activation of proinflammatory
cytokines. Despite the distinction between both processes,
cytokine activators and apoptotic caspases have more similarities
than differences. Finally, the odd one among this family, caspase
14, may be involved in keratinocyte differentiation

3) During the past decade, many proteins were shown to be cleaved
during apoptosis in a caspase-dependent manner. Surveys using 2D-
gel electrophoresis revealed that approximately 100 proteins are
cleaved during apoptosis.(1-3) An important question that has yet
to be resolved is whether these are true and necessary substrates
of caspases, or simple bystanders, indeed, only a few have yet
been demonstrated to be important for the proper process of
apoptosis. Hallmark phenotypes of apoptosis are generally a
result of a single protein being cleaved. Much of the time it is
an inactivation event, but there are a few examples of gain of
function caused by caspase proteolysis.

References (abridged):

1. Otto, A.; Muller, E. C.; Brockstedt, E.; Schumann, M.;
Rickers, A.; Bommert, K.; Wittmann-Liebold, B. J. Protein Chem.
1998, 17, 564

2. Brockstedt, E.; Rickers, A.; Kostka, S.; Laubersheimer, A.;
Dorken, B.; Wittmann-Liebold, B.; Bommert, K.; Otto, A. J. Biol.
Chem. 1998, 273, 28057

3. Gerner, C.; Frohwein, U.; Gotzmann, J.; Bayer, E.; Gelbmann,
D.; Bursch, W.; Schulte-Hermann, R. J. Biol. Chem. 2000, 275,
39018

4. Lazebnik, Y. A.; Kaufmann, S. H.; Desnoyers, S.; Poirier, G.
G.; Earnshaw, W. C. Nature 1994, 371, 346

5. Liu, X.; Zou, H.; Slaughter, C.; Wang, X. Cell 1997, 89, 175

Related Material:

ON THE BIOCHEMISTRY OF APOPTOSIS

Nature 2000 407:770

The following points are made by Michael O. Hengartner:

1) Multicellular animals often need to get rid of cells that are
in excess, in the way, or potentially dangerous. To this end,
they use an active dedicated molecular program. As important as
cell division and cell migration, regulated (or programmed) cell
death allows the organism to tightly control cell numbers and
tissue size, and to protect itself from rogue cells that threaten
homeostasis.

2) Discovered and rediscovered several times by various
developmental biologists and cytologists, programmed cell death
acquired a number of names over the past two centuries(1). The
term finally adopted is apoptosis, coined by Currie and
colleagues in 1972 to describe a common type of programmed cell
death that the authors repeatedly observed in various tissues and
cell types(2). The authors noticed that these dying cells shared
many morphological features, which were distinct from the
features observed in cells undergoing pathological, necrotic cell
death, and they suggested that these shared morphological
features might be the result of an underlying common, conserved,
endogenous cell death programme(3).

3) Most of the morphological changes that were observed by Kerr
et al . are caused by a set of cysteine proteases that are
activated specifically in apoptotic cells. These death proteases
are homologous to each other, and are part of a large protein
family known as the caspases(4). Caspases are highly conserved
through evolution, and can be found from humans all the way down
to insects, nematodes and hydra(5). Over a dozen caspases have
been identified in humans; about two-thirds of these have been
suggested to function in apoptosis.

4) All known caspases possess an active-site cysteine, and cleave
substrates at Asp-xxx bonds (that is, after aspartic acid
residues); a caspase's distinct substrate specificity is
determined by the four residues amino-terminal to the cleavage
site. Caspases have been subdivided into subfamilies based on
their substrate preference, extent of sequence identity and
structural similarities.

5) Because they bring about most of the visible changes that
characterize apoptotic cell death, caspases can be thought of as
the central executioners of the apoptotic pathway. Indeed,
eliminating caspase activity, either through mutation or the use
small pharmacological inhibitors, will slow down or even prevent
apoptosis. Thus, blocking caspases can rescue condemned cells
from their apoptotic fate -- a fact that has not escaped the
notice of the pharmaceutical industry.

References (abridged):

1. Vaux, D. L. & Korsmeyer, S. J. Cell death in development. Cell
96, 245-254 (1999)

2. Kerr, J. F., Wyllie, A. H. & Currie, A. R. Apoptosis: a basic
biological phenomenon with wide-ranging implications in tissue
kinetics. Br. J. Cancer 26, 239-257 (1972)

3. Wyllie, A. H., Kerr, J. F. & Currie, A. R. Cell death: the
significance of apoptosis. Int. Rev. Cytol. 68, 251-306 (1980)

4. Alnemri, E. S. et al. Human ICE/CED-3 protease nomenclature.
Cell 87, 171 (1996)

5. Budihardjo, I., Oliver, H., Lutter, M., Luo, X. & Wang, X.
Biochemical pathways of caspase activation during apoptosis.
Annu. Rev. Cell Dev. Biol. 15, 269-290 (1999)

Related Material:

APOPTOSIS AND CASPASES

Nature 2000 403:29

The following points are made by Huseyin Mehmet:

1) In the normal workings of the cell, apoptosis needs to be
tightly regulated by cellular mechanisms in order to avoid
unnecessary cell death and serious pathology (such as possibly
certain neurodegenerative diseases). One way in which the
prevention of unnecessary cell death is accomplished by the cell
is the physical separation of the various components of the
apoptotic machinery, so that only when the "death switch" is
actually tripped are the components involved in apoptosis brought
together and the suicide program activated. The two main cell
compartments now known to be involved in apoptosis are the plasma
membrane, where both death and survival receptors are located,
and the mitochondria of cells, where several proteins that
regulate apoptosis reside.

2) Among the most prominent apoptosis-specific enzymes are the
caspases, a family of cysteine-dependent aspartate-specific
proteases. These enzymes can be broadly divided into two groups:
a) initiator caspases (e.g., caspase-8 and caspase-9) whose main
function is activate other caspases; and b) executor caspases
(e.g., caspase-3, -6, and -7), which are responsible for
dismantling cellular proteins. The two main apoptotic pathways --
the death receptor pathway and the mitochondrial pathway -- are
activated by caspase-8 and caspase-9, respectively, both of which
are found in the cytoplasm. Caspase-8 is recruited by an
apoptosis-inducing signaling complex only when death receptors
are oligomerized after binding of specific ligands. In contrast,
caspase-9 is activated when cytochrome-c is released into the
cytoplasm from the space between the inner and outer
mitochondrial membranes.

3) T. Nakagawa et al (Nature 403:99 2000) have demonstrated that
another caspase involved in apoptosis, caspase-12, is localized
in the endoplasmic reticulum. Caspase-12 is apparently
specifically involved in apoptosis that results from various
biochemical stresses to the endoplasmic reticulum, such as
disruption of endoplasmic reticulum calcium ion distributions.
Apoptosis triggered through pathways that do not involve the
endoplasmic reticulum apparently does not result in the
activation of caspase-12.

4) The author concludes that since caspases are central to both
normal programmed cell death and injury-dependent apoptosis, any
therapy that manipulates caspase activity must take into account
the possible effects on tissue viability. "If activation of
caspase-12 does turn out to be confined to only a narrow band of
cellular stress signals, it will be a promising potential target
for treating neurodegenerative diseases and cancer with few side
effects."

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3. MITOCHONDRIA AND APOPTOSIS

APOPTOSIS, MITOCHONDRIA, AND CASPASES

Science 1998 281:1309,1312

The following points are made by D.R. Green and J.C. Reed:

1) The current consensus among biologists is that approximately 2
billion years ago the cells destined to become the ancestors of
all eukaryotes entered into a partnership with an ancestor of
today's *purple bacteria, an ancestor that subsequently became
the mitochondria of today.

2) It has been hypothesized by several investigators that the
*endosymbiotic origins of mitochondria and the evolution of
aerobic metabolism in eukaryotes formed the basis for the
evolution of active cell death, which in metazoans is manifested
predominantly as apoptosis. Central roles for mitochondria as the
orchestrators of apoptosis have been firmly established in many
systems.

3) In recent years it has become apparent that the effectors of
apoptosis are a family of intracellular proteases known as
"caspases", although inhibiting these enzymes does not always
prevent apoptosis.

4) At least 3 general mechanisms have been proposed for the
involvement of mitochondria in the control of cell life and
death: a) disruption of *electron transport, *oxidative
phosphorylation, and adenosine triphosphate (ATP) production; b)
release of proteins that trigger activation of the caspases
family of proteases; c) alteration of cellular *redox potentials.

5) In many apoptosis scenarios, the mitochondrial inner
electrical transmembrane potential collapses, indicating the
opening of large conductance channels through the inner membrane.
In contrast, certain stimuli can induce rupture of the outer
membrane of mitochondria and release of caspase-activation
proteins. The authors conclude: "Perhaps a few hundred million
years ago, either convergent or divergent evolutionary processes
allowed the ... fundamental framework for bacterial warfare to be
incorporated into the cell death mechanisms used by animal cells,
thereby establishing mitochondria as important participants not
only in animal cell life but also in active cell death."

In a companion and contiguous review of caspases and apoptosis,
N.A. Thornberry and Y. Lazebnik point out the following:

1) Proteolysis is irreversible, which implies that regulation of
proteases is limited to control of their activity and
availability of substrate -- the only known way of "correcting" a
cleaved protein is to make it afresh.

2) Most proteases are synthesized as precursors that have little
if any catalytic activity. The precursor is usually converted to
the active enzyme by proteolytic processing mediated either by
another protease or by autocatalysis. Thus large amounts of
precursor can be accumulated in advance and activated on demand.

3) Proteases can regulate their own activation, resulting in an
exponential rate of activation.

4) Where there are proteases there are inhibitors, and these
inhibitors regulate the concentration of active protease in the
cell.

5) Proteolytic reactions can be specific, determined by a
combination of primary, secondary, or tertiary structures of
protein substrates. Proteolysis that governs critical biological
processes such as the cell cycle or cell death is highly specific
and involves a restricted set of substrates.

6) The various caspases share similarities in amino acid
sequence, structure, and substrate specificity.

7) Caspases are among the most specific of proteases with an
unusual and absolute requirement for cleavage after aspartic acid
and recognition of at least 4 amino acids terminal to the
cleavage site.

8) The strict specificity of caspases is consistent with the
observation that apoptosis is not accompanied by indiscriminate
protein digestion, but rather a select set of proteins is cleaved
in a coordinated manner, usually at a single site, resulting in a
loss or change in function.

9) Apoptotic events include DNA fragmentation, chromatin
condensation, membrane blebbing, cell shrinkage, and disassembly
into membrane-enclosed vesicles (apoptotic bodies). In vivo, this
process culminates with the engulfment of apoptotic bodies by
other cells, preventing complications that would result from a
release of intracellular contents. In apoptosis, these changes
occur in a predictable reproducible sequence and can be completed
with 30 to 60 minutes. The authors conclude: "Substantial
progress has been made in understanding the structural and
catalytic properties of active caspases and their contribution to
apoptosis. The goal for future research is to understand the
regulation of these enzymes. This should facilitate efforts to
rationally manipulate the apoptotic machinery for therapeutic
gain."

Notes:

*purple bacteria: Specifically, any of the various photosynthetic
bacteria that contain bacteriochlorophyll, and are thus
distinguished by purplish or reddish-brown pigments. But the term
"purple bacteria" is sometimes used as a synonym for the phylum
Proteobacteria, a general category comprising a large number of
diverse forms.

*endosymbiotic: Endosymbiosis is an arrangement in which one
organism lives inside another organism, but the term is usually
restricted to arrangements of mutual benefit, thus not including
parasite-host relationships. A number of eukaryotic cell
organelles (including mitochondria) are believed to have
originated from endosymbiotic relationships between eukaryotic
cells and simpler cells.

*oxidative phosphorylation: Production of ATP during aerobic
respiration. It takes place in the mitochondria of eukaryotic
cells and requires molecular oxygen as a terminal electron
acceptor.

*redox potentials: Chemical potentials in a chemical reaction
involving the simultaneous reduction and oxidation of two
compounds by a transfer of electrons between them.

*chromatin: The entire complex of a eukaryotic chromosome,
including DNA, chromosomal proteins, and chromosomal RNA.

Related Material:

APOPTOSIS AND CALCIUM

Science 2003 300:65

The following points are made by N. Demaurex and C. Distelhorst:

1) The cells of our body are able to quickly commit suicide in
response to genetic or environmental cues, a process termed
apoptosis. This process is essential for development, tissue
homeostasis, and defense against pathogens. Organized life
requires cell death, and execution of cell death relies on the
very machinery of life.

2) Mitochondria, the organelles that produce energy through
cellular respiration, integrate death signals mediated by
proteins belonging to the Bcl-2/Bax family, and kill cells by
releasing critical factors such as cytochrome c that activate
executioner caspase proteases (1,2). Calcium ions (Ca2+), the
cellular messengers that control every aspect of cell and tissue
physiology, can be turned into death signals when delivered at
the wrong time and place (3,4). Mitochondria eventually decide
whether Ca2+ signals are decoded as life or death signals (5),
but it is not clear whether Ca2+ is an additional stress factor
that "tips the balance" or is an obligatory signal for death.

3) Scorrano et al. (Science 2003 300:135) have demonstrated that
the transfer of Ca2+ from the endoplasmic reticulum (ER) to the
mitochondria is required for initiation of programmed cell death
by some, but not all, apoptotic signals. Their approach of
genetically inactivating crucial proteins and reconstituting them
in specific organelles reveals that the Ca2+ content of the ER
determines the cell's ability to commit suicide, defining the ER
as a new gateway to apoptosis.

References (abridged):

1. D. R. Green, J. C. Reed, Science 281, 1309 (1998)

2. J. C. Martinou, D. R. Green, Nature Rev. Mol. Cell. Biol. 2,
63 (2001)

3. M. J. Berridge, P. Lipp, M. D. Bootman, Nature Rev. Mol. Cell.
Biol. 1, 11 (2000)

4. G. Hajnoczky, G. Csordas, M. Madesh, P. Pacher, Cell Calcium
28, 349 (2000)

5. G. Szalai, R. Krishnamurthy, G. Hajnoczky, EMBO J. 18, 6349
(1999)

Related Material:

APOPTOSIS, MITOCHONDRIA, AND CHEMICAL GENETICS

Science 2003 299:214

The following points are made by D.W. Nicholson and N.A.
Thornberry:

1) Screening small molecules for their ability to perturb a
cellular pathway, and subsequently identifying the specific
targets of the active compounds, is a powerful way to study
complex biological processes. This approach, known as "forward
chemical genetics" (1), may lead to the identification of new
regulatory mediators of biochemical pathways, and/or validation
of molecular targets for therapeutic intervention. Regarding the
latter, there is the added virtue that the chemical tractability
of the putative target can be established. A twist on this theme,
"reverse pharmacology," enables identification of the molecular
targets of efficacious pharmacological agents with unknown
mechanisms of action.

2) Mammalian cells contain sophisticated machinery that permits
them to quickly commit suicide in response to physiological,
pathogenic, or cytotoxic stimuli. The overwhelming majority of
cell death signals engage the mitochondrial (or "intrinsic")
pathway in which the proteolytic enzyme caspase-9 is recruited
and activated. Activation of caspase-9 is mediated by a
macromolecular complex, the "apoptosome", that is formed in
response to a cellular commitment to apoptotic. Formation of the
apoptosome is initiated upon release of certain mitochondrial
proteins, such as cytochrome c, from the mitochondrial
intermembrane space. Released cytochrome c binds to monomers of
Apaf-1 in the cytosol, inducing a conformational change that
enables stable association with (deoxy)adenosine triphosphate.
Apaf-1 monomers then assemble into the heptameric apoptosome,
which in turn binds to procaspase-9. Once recruited, procaspase-9
acquires catalytic competency, is proteolytically cleaved, and
activates the effector caspases (caspase-3, -6, and -7), a
process that culminates in apoptotic cell death.

3) Although typically applied to whole cell assays, chemical
genetics is equally valuable for identifying mediators of
cellular processes in relevant in vitro assays. One such process
is the mitochondrial pathway of programmed cell death
(apoptosis), identified and reconstituted in vitro by Wang and
colleagues in 1997 (2-4). X. Jiang et al (5) apply chemical
genetics to their reconstituted in vitro cell death assay. In so
doing, they identify a small-molecule activator of apoptosis, and
implicate two proteins with links to cancer pathogenesis in the
regulation of mitochondrial cell death.

References (abridged):

1. B. R. Stockwell, Nature Rev. Genet. 1, 116 (2000)

2. X. Liu et al., Cell 86, 147 (1996)

3. H. Zou et al., Cell 90, 405 (1997)

4. P. Li et al., Cell 91, 479 (1997)

5. X. Jiang et al., Science 299, 223 (2003)

Related Material:

APOPTOSIS AND MITOCHONDRIAL MEMBRANE PERMEABILIZATION

Current Biology 2003 13:R71

The following points are made by N. Zamzami and G. Kroemer:

1) The point-of-no-return of apoptotic cell death is mostly
determined by two intertwined phenomena, namely mitochondrial
membrane permeabilization (MMP) and caspase activation [1]. MMP
culminates in the complete loss of the barrier function of the
outer mitochondrial membrane and the consequent release of
potentially toxic proteins that are normally secured in the
intermembrane space between the inner and outer mitochondrial
membranes. Such cytotoxic proteins include caspase-independent
death effectors (nucleases and proteases) as well as caspase
activators, namely cytochrome c (which activates the so-called
apoptosome, a caspase activation complex containing Apaf-1 and
pro-caspase-9), and Smac/DIABLO and Omi/Htr2 (which both block
the AIPfamily of caspase inhibitors).

2) A plethora of different pro-apoptotic signals trigger MMP. One
such stimulus is provided by caspase-8, which mediates the
proteolytic maturation of the pro-apoptotic Bcl-2 family protein
Bid. Caspase-8-digested Bid (also called "truncated Bid", t-Bid)
translocates from the cytosol to mitochondrial membranes,
attracted by the mitochondrion-specific lipid cardiolipin [2].
Bid causes MMP in a manner that is strictly dependent on the
presence of either of two pro-apoptotic Bcl-2 proteins, namely
Bax or Bak, as indicated by mouse knock-out studies [3]. The t-
Bid-triggered Bax-dependent MMP correlates with the full
insertion of Bax in the membrane, linked to a conformational
change with exposure of the amino terminus, and the formation of
Bax oligomers [4]. The apoptosis-inhibitory oncoproteins Bcl-2
and Bcl-xL, which are predominantly present in mitochondrial
membranes, inhibit apoptosis by locally antagonizing Bax or Bak
[5]. It thus appears that pro- and anti-apoptotic members of the
Bcl-2 family locally engage in a battle of molecular interactions
to regulate MMP.

3) In summary: One critical step of apoptosis is the release of
mitochondrial proteins through the outer mitochondrial membrane.
Recent work shows that two pro-apoptotic Bcl-2 family proteins,
Bax and Bid, as well as the mitochondrion-specific lipid
cardiolipin may cooperate in chemically defined liposomes to
generate a protein-permeable conduit, relaunching the debate on
the identity of the pore responsible for mitochondrial membrane
permeabilization during apoptosis.

References (abridged):

1. Kroemer, G. and Reed, J.C. (2000). Mitochondrial control of
cell death. Nat. Med. 6, 513-519

2. Lutter, M., Fang, M., Luo, X., Nishijima, M., Xie, X.S., and
Wang, X. (2000). Cardiolipin provides specificity for targeting
tBid to mitochondria. Nat. Cell Biol. 2, 754-756

3. Wei, M.C., Zong, W.-X., Cheng, E.H.-Y., Lindsten, T.,
Panoutsakopoulou, V., Ross, A.J., Roth, K.A., MacGregor, G.R.,
Thompson, C.B., and Korsmeyer, S.J. (2001). Proapoptotic BAX and
BAK: A requisite gateway to mitochondrial dysfunction and death.
Science 292, 727-730

4. Eskes, R., Desagher, S., Antonsson, B., and Martinou, J.C.
(2000). Bid induces the oligomerization and insertion of Bax into
the outer mitochondrial membrane. Mol. Cell. Biol. 20, 929-935

5. Letai, A., Bassik, M., Walensky, L., Sorcinelli, M., Weiler,
S., and Korsmeyer, S. (2002). Distinct BH3 domains either
sensitize or activate mitochondrial apoptosis, serving as
prototype cancer therapeutics. Cancer Cell. 2, 183

Related Material:

APOPTOSIS AND MITOCHONDRIA

Current Biology 2002 12:R177

The following points are made by B.B. Wolf and D.R. Green:

1) Apoptosis is a conserved cellular suicide program that
eradicates excess or potentially dangerous cells. Its many
important physiological functions include terminating immune
responses and eliminating virally infected or cancerous cells
[1]. The induction of apoptosis relies critically on the
mitochondria, as these organelles release apoptogenic factors
that activate caspases, a family of proteinases that kill the
cell via proteolysis of key substrates [2]. Recent studies [3 5]
have shed new light on this process and shown that, during cell
death, mitochondria release a protein known as Omi into the
cytosol. In the cytosol, Omi augments caspase activation by
blocking endogenous caspase inhibitors and may also activate
caspase-independent death pathways through its serine protease
activity.

2) Two main pathways have been defined that initiate caspase
activation [2]. The first begins at the cell surface and involves
ligand-induced activation of death receptors, such as TNFRI and
Fas, which then recruit and activate caspases. The second
involves mitochondrial integration of cellular stress signals and
subsequent release into the cytosol of cytochrome c, which
activates caspases via the adapter molecule "apoptotic protease
activating factor-1" (APAF-1). In certain cell types (Type II
cells), the induction of apoptosis appears to require
amplification by mitochondria of the initial death receptor
signal. Mitochondria thus play a critical part in the induction
of apoptosis by cellular stress and often by death receptors.

3) In summary: Recent studies have shown that, during cell death,
the protein Omi is released from the mitochondrial intermembrane
space into the cytosol, where it augments caspase-dependent
apoptosis by blocking inhibitors and may induce caspase-
independent cell death via its serine protease activity.

References (abridged):

1. Green, D.R. (2000). Apoptotic pathways: paper wraps stone
blunts scissors. Cell 102, 1-4

2. Wolf, B. and Green, D.R. (1999). Suicidal tendencies:
apoptotic cell death by caspase family proteinases. J. Biol.
Chem. 274, 20049-20052

3. Suzuki, Y., Imai, Y., Nakayama, H., Takahashi, K., Takio, K.,
and Takahashi, R. (2001). A serine protease, HtrA2, is released
from mitochondria and interacts with XIAP, inducing cell death.
Mol. Cell 8, 613-621

4. Martins, L.M., Iaccarino, I., Tenev, T., Gschmeissner, S.,
Totty, N.F., Lemoine, N.R., Savopoulos, J., Gray, C.W., Creasy,
C.L., Dingwall, C. and Downward, J. (2001). The serine protease
Omi/HtrA2 regulates apoptosis by binding XIAP through a reaper-
like motif. J. Biol. Chem. in press.

5. Verhagen, A.M., Silke, J., Ekert, P.G., Pakusch, M., Kaufman,
H., Connolly, L.M., Day, C.L., Tikoo, A., Burke, R. and Wrobel,
C. (2001). HtrA2 promotes cell death through its serine protease
activity and its ability to antagonize inhibitor of apoptosis
proteins. J. Biol. Chem., in press.

Related Material:

MITOCHONDRIA AND APOPTOSIS

The Scientist 2001 25 June

The following points are made by Laura DeFrancesco:

1) There is some evidence that cytochrome c released from
mitochondria could be involved in apoptosis, and some researchers
believe that mitochondria-mediated apoptosis is central in a
number of major debilitating diseases, including Parkinson's
disease and cancer. One theory proposes that cytochrome c release
is mediated by a permeability transition that results from severe
oxidative stress. When cells are stressed, either by injury or by
certain toxins, mitochondrial swelling occurs, which can lead to
the rupture of the outer mitochondrial membrane and the release
of protein from the inter-membrane space. Such a permeability
transition also collapses the mitochondrial membrane potential,
which is vital to ATP production through the respiratory chain.

2) But whether this scenario would produce apoptosis rather than
necrosis is problematic. Dave Nicholls, a researcher in
mitochondria, says: "The field is new, confusing and
contradictory. It's different from a mature field where there is
total consensus. Here there's total confusion -- confusion
because there are multiple forms of apoptosis, many models, and
some of the techniques [are] not being used appropriately."

Related Material:

THE MITOCHONDRIAL MEMBRANE POTENTIAL IN APOPTOSIS

Apoptosis 2003 8:115

The following points are made by J.D.Ly et al:

1) Mitochondrial dysfunction has been shown to participate in the
induction of apoptosis and has even been suggested to be central
to the apoptotic pathway. Indeed, opening of the mitochondrial
permeability transition pore has been demonstrated to induce
depolarization of the transmembrane potential (Deltapsi(m)),
release of apoptogenic factors and loss of oxidative
phosphorylation.

2) In some apoptotic systems, loss of Deltapsi(m) may be an early
event in the apoptotic process. However, there are emerging data
suggesting that, depending on the model of apoptosis, the loss of
Deltapsi(m) may not be an early requirement for apoptosis, but on
the contrary may be a consequence of the apoptotic-signaling
pathway. Furthermore, to add to these conflicting data, loss of
Deltapsi(m) has been demonstrated to not be required for
cytochrome c release, whereas release of apoptosis inducing
factor AIF is dependent upon disruption of Deltapsi(m) early in
the apoptotic pathway.

3) Together, the existing literature suggests that depending on
the cell system under investigation and the apoptotic stimuli
used, dissipation of Deltapsi(m) may or may not be an early event
in the apoptotic pathway. Discrepancies in this area of apoptosis
research may be attributed to the fluorochromes used to detect
Deltapsi(m). Differential degrees of sensitivity of these
fluorochromes exist, and there are also important factors that
contribute to their ability to accurately discriminate changes in
Deltapsi(m).

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4. APOPTOSIS IN DEVELOPMENT

ON THE PROCESS OF APOPTOSIS IN DEVELOPMENT

Nature 2000 407:796

The following points are made by P. Meier et al:

1) In animal development: during the ontogeny of many organs,
cells are over-produced only to be etched or whittled away to
generate the rococo structures of functional tissues. Early
distaste among biologists for the "wastefulness" of such a
process has given away to the recognition that the ability to
ablate cells is as essential a constructive process in animal
ontogeny as are the abilities to replicate and differentiate
them. After all, most animals thrive in a sea of energy and
profligacy with their component cells is a small price to pay for
ability to move around and propagate. It is highly unlikely that
the peacock, upon encountering the peahen of his dreams, demurs
to ponder the energetic cost of his outrageous tail.

2) It is now clear that physiological cell death is an essential
component of animal development, important for establishment and,
in vertebrates at least, maintenance of tissue architecture. A
general modus operandi of metazoan development is the over-
production of excess cells followed by an apoptotic culling
during later stages of development to match the relative number
of cells of different types to achieve proper organ function(1).
Thus, during animal development, numerous structures are formed
that are later removed by apoptosis. This enables greater
flexibility as primordial structures can be adapted for different
functions at various stages of life or in different sexes. Thus,
the M�llerian duct gives rise to the uterus and oviduct in
females but it is not needed in males and so is consequently
removed. On the other hand, the Wolffian duct is the source of
male reproductive organs and is deleted in females. Organisms are
like many modern computer programs, full of remnant code that was
once used in an ancestral incarnation or that runs irrelevant
routines that nobody needs. During development, apoptosis is
frequently used to expunge such structures. For instance, early
in vertebrate development, the pronephric kidney tubules arise
from the nephrogenic mesenchyme. Although these pronephric
tubules form functioning kidneys in fish and in amphibian larvae,
they are not active in mammals and degenerate(2). Similarly,
during insect and amphibian metamorphosis, apoptosis ablates
cells that are no longer needed such as muscles and neurons
essential for larval locomotion in insects or the amphibian
tadpole tail.

3) Apoptosis also acts as part of a quality-control and repair
mechanism that contributes to the high level of plasticity during
development by compensating for many genetic or stochastic
developmental errors. For example, Drosophila embryos with extra
doses of the morphogen bicoid (bcd) gene show severe
mispatterning in their anterior regions. Surprisingly, these
embryos develop into relatively normal larvae and adults because
cell death compensates for tissue overgrowth and
mispatterning(3). Cells that have been incorrectly programmed
are, in effect, misplaced cells. They therefore fail to receive
the appropriate trophic signals for their survival and
consequently activate their innate auto-destruct mechanism.(4,5)

References (abridged):

1. Jacobson, M. D., Weil, M. & Raff, M. C. Programmed cell death
in animal development. Cell 88, 347-354 (1997)

2. Sax‚n, L. Organogenesis of the Kidney (Cambridge Univ. Press,
Cambridge, 1987)

3. Namba, R., Pazdera, T. M., Cerrone, R. L. & Minden, J. S.
Drosophila embryonic pattern repair: how embryos respond to
bicoid dosage alteration. Development 124, 1393-1403 (1997)

4. Thornberry, N. A. & Lazebnik, Y. Caspases: enemies within.
Science 281, 1312-1316 (1998)

5. Hengartner, M. Apoptosis. Death by crowd control. Science 281,
1298-1299 (1998)

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5. APOPTOSIS IN THE NERVOUS SYSTEM

ON NEURON DEATH IN THE NERVOUS SYSTEM

Nature 2000 407:802

The following points are made by J. Yuan and B.A. Yankner:

1) Although mature neurons are among the most long-lived cell
types in mammals, immature neurons die in large numbers during
development. Furthermore, neuronal cell death is the cardinal
feature of both acute and chronic neurodegenerative diseases. How
do neurons die? This is a difficult question and we have only
recently begun to understand the basic mechanisms.

2) Like all cells, neuronal survival requires trophic support.
Viktor Hamburger and Rita Levi-Montalcini described in a seminal
paper that the survival of developing neurons is directly related
to the availability of their innervating targets(1). This laid
the foundation for the neurotrophin hypothesis(2), which proposed
that immature neurons compete for target-derived trophic factors
that are in limited supply; only those neurons that are
successful in establishing correct synaptic connections would
obtain trophic factor support to allow their survival. The
neurotrophin hypothesis predicts correctly that neuronal survival
requires a positive survival signal; it did not, however, provide
a concrete hypothesis as to how neurons die in the absence of
trophic support.

3) It was assumed until recently that neurons die simply of
passive starvation in the absence of trophic factors. In 1988,
using cultured sympathetic neurons as a model system, Johnson and
colleagues showed that inhibition of RNA and protein synthesis
blocked sympathetic neuronal cell death induced by nerve growth
factor (NGF) deprivation(3), providing the first tangible
evidence that neurons might actually instigate their own demise.
The identification of the programmed cell death genes ced-3, ced-
4 and ced-9, in the nematode Caenorhabditis elegans and their
mammalian homologues, opened a window of opportunity to examine
the mechanism of neuronal cell death at the molecular level(4).
It was soon discovered that vertebrate neuronal cell death
induced by trophic factor deprivation requires the participation
of cysteine proteases, later termed "caspases", which are the
mammalian homologues of the C. elegans cell death gene product
CED-3 (5). This was the first functional evidence that trophic
factor deprivation activates a cellular suicide program in
vertebrate neurons. What are the critical components of this
neuronal suicide program? How is it activated by lack of trophic
support during development and by pathological conditions in
neurodegenerative diseases? These questions have been studied
intensively during the past decade.

References (abridged):

1. Hamburger, V. & Levi-Montalcini, R. J. Exp. Zool. 111, 457-502
(1949)

2. Purves, D. Body and Brain: A Trophic Theory of Neural
Connections (Harvard Press, Cambridge, Massachusetts, 1988)

3. Martin, D. P. et al. Inhibitors of protein synthesis and RNA
synthesis prevent neuronal death caused by nerve growth factor
deprivation. J. Cell Biol. 106, 829-844 (1988)

4. Metzstein, M. M., Stanfield, G. M. & Horvitz, H. R. Genetics
of programmed cell death in C. elegans: past, present and future.
Trends Genet. 14, 410-416 (1998)

5. Gagliardini, V. et al. Prevention of vertebrate neuronal death
by the crmA gene. Science 263, 826-828 (1994)

Related Material:

AXONAL SELF-DESTRUCTION AND NEURODEGENERATION

Science 2002 296:868

The following points are made by M.C. Raff et al:

1) Much effort is being devoted to understanding the nature of
neuronal cell death in various neurodegenerative diseases such as
motor neuron disease, glaucoma, and Alzheimer, Parkinson, and
Huntington diseases (1-5). It may be, however, that neuronal
death in these diseases occurs too late to be clinically
important. Degeneration of the neuron's long process -- the axon
-- often precedes the death of the cell body and may make a more
important contribution to the patient's disability.

2) A classical example of axonal degeneration is "Wallerian
degeneration", which occurs when an axon is cut. The part of the
axon that is now disconnected from the cell body disassembles in
a characteristic and orderly way. In vertebrates, this part of
the axon can continue to conduct action potentials for a day or
two when electrically stimulated, but it then quickly
degenerates: The endoplasmic reticulum breaks down, the
neurofilaments degrade, the mitochondria swell, and the axon
breaks up into fragments that are phagocytosed. Wallerian
degeneration can occur in both the peripheral nervous system
(PNS) and central nervous system (CNS) whenever trauma, a
vascular accident, infection, or an immune response locally
injures axons.

3) From the perspective of neurodegenerative diseases, a more
relevant form of axonal degeneration occurs in a process called
"dying back". Here, the axon of an unhealthy neuron progressively
degenerates over weeks or months, beginning distally and
spreading toward the cell body. This is the most common pathology
seen in peripheral nerve diseases caused by a wide variety of
toxic, metabolic, and infectious insults. It occurs, for example,
in the polyneuropathies associated with diabetes, alcoholism,
acrylamide poisoning, and AIDS. It also seems to occur in CNS
neurodegenerative diseases, including motor neuron disease and
Alzheimer and Parkinson diseases, although it has been less well
documented in these cases. The dying-back process is intriguing:
How can a cell eliminate part of itself while leaving the rest
intact? It contrasts with Wallerian degeneration, in which the
axonal lesion itself both starts the destructive process and
compartmentalizes it.

4) In summary: Neurons seem to have at least two self-destruct
programs. Like other cell types, they have an intracellular death
program for undergoing apoptosis when they are injured, infected,
or not needed. In addition, they apparently have a second,
molecularly distinct self-destruct program in their axon. This
program is activated when the axon is severed and leads to the
rapid degeneration of the isolated part of the cut axon. Do
neurons also use this second program to prune their axonal tree
during development and to conserve resources in response to
chronic insults?

References (abridged):

1. C. Behl, J. Neural Transm. 107, 1325 (2000)

2. R. M. Gibson, Br. Med. J. 322, 1539 (2001)

3. N. Heintz and H. Y. Zoghbi, Annu. Rev. Physiol. 62, 779 (2000)

4. L. J. Martin, Int. J. Mol. Med. 7, 455 (2001)

5. N. N. Osborne, et al., Br. J. Ophthalmol. 83, 980 (1999)

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6. APOPTOSIS AND CLINICAL MEDICINE

ON APOPTOSIS-BASED THERAPEUTIC AGENTS

Nature 2000 407:810

The following points are made by Donald W. Nicholson:

1) The therapeutic landscape has witnessed major changes during
the past few decades. Antisense-based therapies have been made
more viable by better oligonucleotide chemistry, resulting in
increased metabolic stability, cell penetration and fewer side
effects. Injectable recombinant biologicals are demonstrating
clinical efficacy and are becoming more broadly accepted as
legitimate therapeutic agents. Last but not least, combinatorial
chemistry and rapid analogue synthesis -- techniques by which
large numbers of chemical permutations can be quickly assembled -
- have greatly accelerated the pace at which potential drug
candidates can be generated. These remarkable accomplishments
have positioned the apoptosis field for what is hoped will be a
smooth transition from bench to clinic.

2) Even a quick glance at the molecular components of the cell
death pathway reveals many opportunities for apoptosis
modulation. With this plethora of opportunities unfortunately
comes a number of practical limitations, not the least of which
is the practical issue that the cell death pathway as currently
known contains very few conventional drug targets, such as
enzymes and small-ligand receptors. Attention has therefore
focused on other strategies to affect the proteinaceous
components of the apoptotic pathway.

3) Modulating the expression of key molecular components of the
cell death machinery is an attractive and obvious strategy. But
whereas gene and antisense therapy seem the most viable
approaches at present to alter gene expression, and antisense
Bcl-2 shows promise in the treatment of cancer, these
technologies are still in their infancy.

4) An alternative is to interfere with specific protein protein
interactions inside or outside the cell. But despite intensive
research, small molecules that interfere with specific protein
protein interactions are almost unheard of because the surface
areas between interacting polypeptides are large and difficult to
disrupt. The "critical interaction" strategy, where small-
molecule inhibitors disrupt only key sites of binding between
interacting polypeptides, is sound in principal but has been
lacking in practice. Even so, small molecules with a high
affinity for the binding cleft of the Bcl-2 homology (BH) domain
BH3 on the surface of Bcl-2 seem to induce apoptosis in cultured
cells(2).

5) For recombinant protein strategies, a different set of equally
complex issues arise. For example, there is always the
possibility that the cells of the immune system will develop
autoantibodies against recombinant proteins and, furthermore,
their use is mostly limited to extracellular targets because
large proteins do not readily enter the cell.

6) Opportunities are scarce for the development of pharmaceutical
therapeutics to modulate the apoptotic pathway; the exception
being organic small-molecule inhibitors of caspase activity. But
there is no precedent of human therapeutics that successfully
target cysteine proteases owing to the difficulties in developing
electrophiles that are specific enough that for them not to
attack other biological nucleophiles.(3-5)

References (abridged):

1. Ellis, R. E., Yuan, J. Y. & Horvitz, H. R. Mechanisms and
functions of cell death. Annu. Rev. Cell Biol. 7, 663-698 (1991)

2. Wang, J. L. et al. Structure-based discovery of an organic
compound that binds bcl-2 protein and induces apoptosis of tumor
cells. Proc. Natl Acad. Sci. USA 97, 7124-7129 (2000)

3. Adams, J. M. & Cory, S. The Bcl-2 protein family: arbiters of
cell survival. Science 281, 1322-1326 (1998)

4. Reed, J. C. Bcl-2 family proteins. Oncogene 17, 3225-3236
(1998)

5. Veis, D. J., Sorenson, C. M., Shutter, J. R. & Korsmeyer, S.
J. Bcl-2-deficient mice demonstrate fulminant lymphoid apoptosis,
polycystic kidneys, and hypopigmented hair. Cell 75, 229-240
(1993)

Related Material:

NONINVASIVE REAL-TIME IMAGING OF APOPTOSIS

Proc. Nat. Acad. Sci. 2002 99:16551

The following points are made by B. Laxman et al:

1) A majority of clinical imaging is relegated to obtaining
anatomical information based on differences in physical
parameters to generate image contrast. Significant efforts
recently have focused on developing approaches to use noninvasive
imaging technologies to obtain information related to specific
molecular events. These efforts have been focused on reporting of
gene expression (1-5) or extracellular proteolytic activity by
using synthetic fluorescent probes. However, real-time detection
of a single specific intracellular enzyme or pathway in vivo has
been largely elusive to date.

2) Proteases play a major role in biological processes including
tissue remodeling, vascular hemostasis, digestion, protein
turnover and maturation as well as apoptosis. Apoptosis is a
physiologic process in normal development and homeostasis of
multicellular organisms. Evaluation of therapeutic agents against
pathologies involving an imbalance in apoptosis (e.g., benign
prostate hyperplasia) would greatly benefit from a method to
noninvasively image the specific molecular mediators of
apoptosis. Because cytosolic caspases play a central role in
mediating the initiation and propagation of the apoptotic
cascade, the ability to noninvasively image the activation of
these zymogens would provide an opportunity to evaluate
therapeutic interventions dynamically in living animals.

3) In summary: Strict coordination of proliferation and
programmed cell death (apoptosis) is essential for normal
physiology. An imbalance in these two opposing processes results
in various diseases including AIDS, neurodegenerative disorders,
myelodysplastic syndromes, ischemia/reperfusion injury, cancer,
autoimmune disease, among others. Objective and quantitative
noninvasive imaging of apoptosis would be a significant advance
for rapid and dynamic screening as well as validation of
experimental therapeutic agents. The authors report the
development of a recombinant luciferase reporter molecule that
when expressed in mammalian cells has attenuated levels of
reporter activity. In cells undergoing apoptosis, a caspase-3-
specific cleavage of the recombinant product occurs, resulting in
the restoration of luciferase activity that can be detected in
living animals with bioluminescence imaging. The ability to image
apoptosis noninvasively and dynamically over time provides an
opportunity for high-throughput screening of proapoptotic and
antiapoptotic compounds and for target validation in vivo in both
cell lines and transgenic animals.

References (abridged):

1. Gambhir, S. S., et al. (2000) Neoplasia 2, 118-138

2. Weissleder, R., et al. (2000) Nat. Med. 6, 351-355

3. Ponomarev, V., et al. (2001) Neoplasia 3, 480-488

4. Doubrovin, M., et al. (2001) Proc. Natl. Acad. Sci. USA 98,
9300-9305

5. Rehemtulla, A., et al. (2002) Mol. Imaging 1, 43-55

Related Material:

INHIBITING AXON DEGENERATION AND SYNAPSE LOSS ATTENUATES
APOPTOSIS AND DISEASE PROGRESSION IN A MOUSE MODEL OF MOTONEURON
DISEASE

Current Biology 2003 13:669

The following points are made by A. Ferri et al:

1) Apoptosis is a hallmark of motoneuron diseases such as
amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy
(SMA) [1]. In a widely used mouse model of motoneuron disease
(progressive motor neuronopathy or pmn) [2 4], transgenic
expression of the anti-apoptotic bcl-2 gene [5] or treatment with
glial cell-derived neurotrophic factor prevents the apoptosis of
the motoneuron soma; however, these treatments were unable to
affect the life span of the animals.

2) The authors report a study to determine whether the pmn
phenotype could be rescued by means of a gene that inhibits axon
degeneration. For this reason, the pmn mice were crossed with
mice bearing the dominant Wlds ( slow Wallerian degeneration )
mutation, which slows axon degeneration and synapse. The authors
demonstrate that the Wlds gene product attenuates symptoms,
extends life span, prevents axon degeneration, rescues motoneuron
number and size, and delays retrograde transport deficits in
pmn/pmn mice. The authors suggest these results point to new
pathogenic mechanisms and therapeutic avenues for motoneuron
diseases.

References (abridged):

1. Cleveland, D. and Rothstein, J. (2001). From Charcot to Lou
Gehrig: deciphering selective motor neuron death in ALS. Nat.
Rev. Neurosci. 2, 806-819

2. Schmalbruch, H., Jensen, H.J., Bjaerg, M., Kamieniecka, Z.,
and Kurland, L. (1991). A new mouse model with progressive motor
neuronopathy. J. Neuropathol. Exp. Neurol. 50, 192-204

3. Martin, N., Jaubert, J., Gounon, P., Salido, E., Haase, G.,
Szatanik, M., and Guenet, J.L. (2002). A missense mutation in
Tbce causes progressive motor neuronopathy in mice. Nat. Genet.
32, 443-447

4. Bommel, H., Xie, G., Rossoll, W., Wiese, S., Jablonka, S.,
Boehm, T., and Sendtner, M. (2002). Missense mutation in the
tubulin-specific chaperone E (Tbce) gene in the mouse mutant
progressive motor neuronopathy, a model of human motoneuron
disease. J. Cell Biol. 159, 563-569

5. Sagot, Y., Dubois-Dauphin, M., Tan, S.A., de Bilbao, F.,
Aebischer, P., Martinou, J.C., and Kato, A.C. (1995). Bcl-2
overexpression prevents motoneuron cell body loss but not axonal
degeneration in a mouse model of a neurodegenerative disease. J.
Neurosci. 15, 7727-7733

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OVERCOMING RESISTANCE OF CANCER CELLS TO APOPTOSIS

J Cell Physiol 2003 196:9

The following points are made by P. Hersey and X.D. Zhang:

1) Discovery of the B cell lymphoma gene 2 (Bcl-2 gene) led to
the concept that development of cancers required the simultaneous
acquisition, not only of deregulated cell division, but also of
resistance to programmed cell death or apoptosis. Apoptosis is
arguably the common pathway to cell death resulting from a range
of therapeutic initiatives, so that understanding the basis for
the resistance of cancer cells to apoptosis may hold the key to
development of new treatment initiatives.

2) Much has already been learnt about the apoptotic pathways in
cancer cells and proteins regulating these pathways. In most
cells, apoptosis is dependent on the mitochondrial dependent
pathway. This pathway is regulated by pro- and anti-apoptotic
members of the Bcl-2 family, and manipulation of these proteins
offers scope for a number of treatment initiatives. Effector
caspases activated by the mitochondrial pathway or from death
receptor signaling are under the control of the inhibitor of
apoptosis protein (IAP) family. Certain proteins from
mitochondrial can, however, competitively inhibit their binding
to effector caspases.

3) Information about the structure of these proteins has led to
initiatives to develop therapeutic agents to block the IAP
family. In addition to development of selective agents based on
these two (Bcl-2 and IAP) protein families, much has been learned
about signal pathways that may regulate their activity. These in
turn might provide additional approaches based on selective
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CELL DEATH AND IMMUNITY: APOPTOSIS AS AN HIV STRATEGY TO ESCAPE
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Nat Rev Immunol 2003 3:392

The following points are made by M.L.Gougeon:

Viruses have evolved numerous mechanisms to evade the host immune
system and one of the strategies developed by HIV is to activate
apoptotic programs that destroy immune effectors. Not only does
the HIV genome encode pro-apoptotic proteins, which kill both
infected and uninfected lymphocytes through either members of the
tumor-necrosis factor family or the mitochondrial pathway, but it
also creates a state of chronic immune activation that is
responsible for the exacerbation of physiological mechanisms of
clonal deletion. The author discusses the molecular mechanisms by
which HIV manipulates the apoptotic machinery to its advantage,
assessing the functional consequences of this process and
evaluating how new therapeutics might counteract this strategy.

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ON REOVIRUS-INDUCED APOPTOSIS

Apoptosis 2003 8:141

The following points are made by P. Clarke and K.L. Tyler:

1) Reoviruses infect a variety of mammalian hosts and serve as an
important experimental system for studying the mechanisms of
virus-induced injury. Reovirus infection induces apoptosis in
cultured cells in vitro and in target tissues in vivo, including
the heart and central nervous system (CNS).

2) In epithelial cells, reovirus-induced apoptosis involves the
release of tumor necrosis factor (TNF)-related apoptosis-inducing
ligand (TRAIL) from infected cells and the activation of TRAIL-
associated death receptors (DRs) DR4 and DR5. DR activation is
followed by activation of caspase 8, cleavage of Bid, and the
subsequent release of pro-apoptotic mitochondrial factors. By
contrast, in neurons, reovirus-induced apoptosis involves a wider
array of DRs, including TNFR and Fas, and the mitochondria appear
to play a less critical role.

3) These results show that reoviruses induce apoptotic pathways
in a cell and tissue specific manner. In vivo there is an
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Post-mitotic neurons and heart muscle cells undergo apoptotic
cell death in a variety of acute and chronic degenerative
diseases. The intrinsic pathway of apoptosis involves the
permeabilization of mitochondrial membranes, which leads to the
release of protease and nuclease activators, and to bioenergetic
failure. Mitochondrial permeabilization is induced by a variety
of pathologically relevant second messengers, including reactive
oxygen species, calcium, stress kinases and pro-apoptotic members
of the Bcl-2 family. Several pharmacological agents act on
mitochondria to prevent the permeabilization of their membranes,
thereby inhibiting apoptosis. Such agents include inhibitors of
the permeability transition pore complex (in particular ligands
of cyclophilin D), openers of mitochondrial ATP-sensitive or
Ca(2+)-activated K(+) channels, and proteins from the Bcl-2
family engineered to cross the plasma membrane. In addition,
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