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cell death process while others, since they happen to contain a
caspase target motif in their sequence, may be cleaved as bystanders.
The caspase substrates playing an active role in the progression of
apoptosis can be divided in two groups. Firstly, there are apoptosis
regulatory proteins which become activated by cleavage and thereby
act as transducers or amplifi ers that determine the apoptotic
threshold and cell fate. Apart from caspases themselves, this can be
exemplifi ed by the DNase inhibitor ICAD/DFF45 (Inhibitor of
Caspase Activated DNase/DNA fragmentation factor 45) which,
upon cleavage by caspase-3, liberates the active CAD/DFF40
(Caspase Activated DNase/DNA fragmentation factor 40) nucle-
ase that mediates apoptotic internucleosomal DNA fragmentation
[ 6 ]. Correspondingly, a truncated version of the Bcl-2 protein fam-
ily member Bid (BH3 interacting domain death agonist) results
from death receptor signaling and caspase-8 proteolytic activity. In
turn, tBid (truncated Bid) promotes release of mitochondrial pro-
apoptotic factors, including cytochrome c , as a consequence of
destabilized membrane potential [ 7 ]. The majority of substrates
are, however, inactivated by caspases. This group consists of pro-
teins implicated in a wide variety of cellular functions, including
structural components, cell adhesion molecules, transcription fac-
tors, as well as factors involved in DNA repair and processing of
RNA [ 8 ]. Detection of caspase substrate cleavage products by means
of SDS-PAGE is often used to confi rm apoptotic conditions in cul-
tured cells. Depending on the experimental context, it may also be
possible to verify a discrete apoptotic signaling route by this method.
It must be noted, however, that substrates generally are cleaved by
effector caspases and that overlapping cleaving activities between
different caspases is common. Thus, conclusions regarding activity
of a specifi c caspase deduced from substrate proteolysis should be
supported by other methods, such as suppression of caspase expres-
sion by means of RNAi technology. One of the most widely used
apoptotic markers in SDS-PAGE methodology is PARP-1 (Poly
[ADP-ribose] polymerase 1) which is a target of caspase-3 in vivo
but can be cleaved by many caspases in vitro. Disruption of PARP-1
activity by proteolytic cleavage results from a separation of the
amino-terminal DNA binding domain (24 kDa) and the carboxy-
terminal catalytic domain (89 kDa). Since PARP-1 is a nuclear poly
(ADP-ribose) polymerase family member implicated in DNA repair
and especially base excision repair a ceased activity of the protein will
promote apoptosis by disruption of cellular homeostasis in general
and genomic integrity in particular [ 9 , 10 ].Cytokeratin 18 (CK18) is a member of the Type I intermediate
fi lament family, that forms heterotetramers. This protein is widely
expressed in normal single layer epithelial tissues and has also been
found at high levels in some tumor tissues. During apoptosis,
cleavage by caspase-3, -6, -7, and -9, exposes a neoepitope in CK181.4 Detection
of Cytokeratin 18
Cleavage by Flow
Cytometry
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