Page 10 - Academic Press Encyclopedia of Physical Science and Technology 3rd Molecular Biology

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Encyclopedia of Physical Science and Technology EN002G-90 May 17, 2001 20:42

548 Cell Death (Apoptosis)

DR3 and TRAILR2 recruit DISC (that consists of FADD radiation and treatment with ceramide (N-acyl-erythro-
and procaspase-8) via the DD of TRADD. Unlike Fas, sphingosine). In addition, extracts from cells activated by
TNFRI, and DR3, TRAILR1 can transmit apoptotic sig- Fas in the presence of a caspase inhibitor do not cause
nals and activate caspase-8 in FADD-deficient fibroblast DNA fragmentation in the cell-free reaction. Indeed,
cells. This suggests that TRAILR1-mediated apoptosis extracts from proliferating cells, in the presence of
is caspase-8-dependent and FADD-independent, although recombinant active caspase-3, induce nuclear apoptosis,
an unidentified FADD-like adapter molecules cannot be indicating that the factors responsible for apoptotic DNA
ruled out at present. The response mediated by DR6 has fragmentation are downstream of the caspase cascade.
not, as yet, been well characterized. It emerged that these factors form a complex composed
In conclusion, ligand-induced trimerized death re- of heterodimeric proteins of caspase-activated DNase
ceptors activate an apical protease, procaspase-8, via (CAD)/DNA fragmentation factor 40 (DFF40)/caspase-
FADD or an unidentified functional homologue. Acti- activated nuclease (CPAN) and an inhibitor of CAD
vated caspase-8 can sequentially activate effector cas- (ICAD)/DFF45 (discussed in the following).
pases responsible for cleaving a variety of death substrates As described above, if dATP is mixed with extracts from
such as ICAD/DFF45 (discussed below), lamin, fodrin, proliferating cells, the extracts have the ability to induce
and poly(ADP-ribose) polymerase. In addition to the di- nuclear apoptosis mediated by caspase-3 activation in a
rect pathway from initiator caspases to effector caspases cell-free reaction. Using this system, Wang’s group have
through Fas, active caspase-8 can transduce death signals purified the factors responsible for processing procaspase-
into mitochondria through the translocation of caspase-8- 3 and identified three gene products, namely Apaf-1,
cleaved Bid from cytosol to mitochondria. Uptake of tBid cytochrome c and procaspase-9. Thus, biochemical ap-
into mitochondria promotes the release of cytochrome c proaches using cell-free systems for apoptosis has demon-
to the cytosol by unknown mechanisms. Once cytochrome strated several important aspects in the field of apoptosis.
c is released, it can generate active caspase-9 via forma- During apoptosis, biochemical procedures have fre-
tion of the apoptosome. Thus, death receptors appear to quently shown loss of mitochondrial function. In order to
use two different pathways for particular tissues in some clarify the regulations of mitochondrial apoptosis, isolated
situations. The two different pathways ensure the death mitochondria have been used. During the early stages of
signals are amplified and target cells killed. study in this field, the function of mitochondria in apop-
totic processes was overlooked due to no apparent changes
on morphology. However, the observations that Bcl-2 (an
V. CELL-FREE SYSTEM IN APOPTOSIS anti-apoptotic protein) is abundant in the mitochondria,
and that apoptotic processes are often accompanied by
The establishment of cell-free systems has provided us a decrease in the mitochondrial membrane potential and
with detailed insight into the cognate molecular mech- by mitochondrial swelling led to a more detailed inves-
anisms of various cellular functions, including gen- tigation of mitochondrial function during apoptosis. Fur-
eral/specific transcriptional regulations, RNA editing, thermore, apoptogenic factors, such as cytochrome c and
protein synthesis and protein degradation, and overall apoptosis-inducing factor (AIF) that had been discovered
metabolic pathways. The well-established biochemical as a nuclear apoptosis-inducing flavoprotein using a cell-
hallmark of apoptosis is chromosomal DNA degrada- free system, are released from the intermembrane space
tion resulting in multimers of 180 bp-nucleosomal units. of mitochondria into the cytosol to induce apoptosis. Re-
Neither protein- nor RNA-synthesis inhibitors block Fas- cently, another factor secreted from mitochondria during
mediated apoptosis, suggesting that all of the components apoptosis, called Diablo/Smac, has been reported. This
for apoptotic induction through Fas are present in cells in factor suppresses the functions of proteins belonging to
latent forms. the inhibitor-of-apoptosis (IAP) family, inhibiting the pro-
A cell-free system for apoptosis was first established tease activity of caspases including caspase-3, -7 and -9.
by exposing isolated nuclei to various extracts and Celldeathislikelytobeacceleratedasaresultofinhibition
monitoring nucleosomal DNA fragmentation. That is, of IAP. Thus, there appear to be various steps to ensure cell
when isolated nuclei from healthy cells were treated killing. How is the loss of mitochondrial function induced
with extracts from dying cells (but not from growing by the various apoptotic stimuli? For example, Bax (Bcl-
cells), they showed apoptotic features, including nuclear 2-associated X protein), which belongs to a member of
morphology with peripheral condensation of chromatin the pro-apoptotic Bcl-2 family, induces apoptosis. When
and nucleosomal DNA fragmentation. This cell-free added individually to purified mitochondria-free cytosol,
system can be reproduced using the extracts from cells neither mitochondria nor Bax can individually induce the
subjected to different apoptotic stimuli, including UV activation of caspases that lead to nuclear apoptosis. In