Page 11 - 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

Cell Death (Apoptosis) 549

contrast, the addition of both Bax and mitochondria trig- to amino acids at 117 (DEPD 117 ) and 224 (DAVD 224 )
gers the release of cytochrome c from mitochondria to cy- (Fig. 4a). Analyses of point mutations at two sites have in-
tosol, which activates the caspase cascade. In addition, it dicated that the cleavage of ICAD at Asp117 and Asp224
has been shown that Bax directly triggers cytochrome c re- by caspase-3 is crucial for the inactivation of ICAD func-
lease from mitochondria in a cell-free reaction. It has been tion. Interestingly, the inhibitory activity of the ICAD
proposed that Bax may release cytochrome c from mito- with single point mutations at both sites was completely
chondria by modulating voltage-dependent anion chan- resistant to treatment with caspase-3. Among the cas-
nel activity and/or causing limited permeabilization of the pases 1 to 8, caspase-7 also cleaves ICAD in an in vitro
mitochondrial outer membrane although this mechanism assay. However, active caspase-7 translocates from cy-
is still controversial. The apoptotic signals from various tosol to mitochondrial and microsomal fractions, whereas
stimuli mediated by mitochondria should be investigated, active caspase-3 is still present in cytosol during Fas-
dissected, and reproduced by biochemical approaches us- induced apoptosis in mouse liver in vivo, suggesting
ing a cell-free system described here. that ICAD in cytoplasm and nuclei are mainly cleaved
by caspase-3. This is consistent with the observation
that caspase-3-deficient cells undergo apoptosis without
VI. APOPTOTIC DNase, CAD chromosomal DNA fragmentation, although caspase-7 is
AND ITS INHIBITOR, ICAD activated.
ICAD are thermostable at 90 C for 5 min and resistant
◦
A biochemical hallmark of apoptosis is chromosomal to denaturants such as 6 M guanidium hydrochloride, 8 M
DNA degradation. It was early on proposed that one of urea and 0.1% SDS. ICAD specifically inhibit the CAD
several known enzymes, including DNase I, DNase II, activity, but not DNase I and DNase II activities, by bind-
DNase γ , and cyclophilins, contribute to chromosomal ing to it. In addition, ICAD can completely inhibit nuclear
DNA fragmentation during apoptosis, although the molec- apoptosis induced by extracts from Fas-activated cells in
ular mechanism understanding apoptotic DNA fragmen- the presence of an inhibitor of caspase-3. Furthermore,
tation by these DNases was unclear. overexpression of caspase-3-resistant ICAD mutants sup-
Most apoptotic stimuli commit the cells to apoptosis presses apoptotic DNA degradation induced by diverse
throughtheactivationofthecaspasecascade.Asdescribed apoptotic stimuli including death factors, growth factor
in section V, several groups, including our own, have es- starvation, anti-cancer drugs, and γ -irradiation, indicating
tablished a caspase-3-inducible cell-free system, in which that CAD needs to be activated to degrade chromosomal
not only chromosomal DNA in nuclei but also naked plas- DNA in many apoptotic situations.
mid DNA is cleaved by caspase-3-activated cell extracts, Molecular cloning of CAD has revealed that the mouse
andusedittoidentifyaresponsiblefactor(s),designatedas CAD gene encodes a basic protein of 344-amino acids and
CAD,forapoptoticDNAfragmentation.WepurifiedCAD a pI of 9.7. Mouse CAD comprises 14 cysteine residues;
from lymphoid cells using the caspase-3-inducible cell- methionine and cysteine residues at positions 1 and 2, re-
free system. We also noticed that an inhibiting-factor(s), spectively, are removed during the maturation of the pro-
designated as ICAD, is present in the extracts from non- tein (Fig. 4b). Human CAD is composed of 338 amino
apoptotic cells and identified ICAD as a 32-kDa protein. acids and is highly homologous to mouse CAD, with an
Molecular cloning of ICAD has revealed that long-form identity of 75.9% between both primary structures. Eleven
(331 amino acids; ICAD-L) and short-form (265 amino cysteine residues, most of which exist as reduced thiol
acids; ICAD-S) of ICAD, which are generated through groups, are conserved between mouse and human CAD.
alternative splicing of the same messenger RNA (Fig. 4a). The C terminal region of CAD contains a stretch of basic
Independently, other groups have purified a latent form amino acids with the features of a nuclear localization sig-
of CAD consisting of heterodimers (DFF40 and DFF45) nal (Fig. 4b). Active CAD is a very unstable protein and
from human HeLa cells or an active form of CAD named easily aggregates under nonreducing condition, in contrast
CPAN from human Jurkat cells, using a system similar to to the CAD/ICAD-L complex. The stability of CAD is en-
ours. hancedbyadditionofreducingreagentssuchasdithiothre-
Both ICAD-L and -S (ICAD) are acidic proteins with itol (DTT) and reduced glutathione, suggesting that inter-
isoelectric points (pI) of around 4.5. A homology search and/or intra-molecular crosslinking of free thiols in CAD
has shown that ICAD-L has high similarity to human may cause the aggregation and inactivation of CAD func-
DFF45, suggesting that mouse ICAD-L is a counterpart tion. Some DNases are inactivated by reagents that modify
of human DFF45. ICAD-L is ubiquitously expressed free thiols, such as iodoacetamide and N-ethyl maleimide,
in a variety of tissues at the same level as ICAD-S. whereas CAD activity is not inhibited by such reagents,
ICAD carry two caspase-3-cleavage sites corresponding suggesting that free thiols in CAD are not required for its