Genetics of Apoptosis

of cytochrome c in caspase activation in C.elegans. The role of cytochrome c in caspase
activation in Drosophila is also currently unclear. Recent data suggest that while
DARK, the Apaf-1 homolog in fly, is clearly necessary for the activation of the apical
caspase DRONC, cytochrome c is not released from mitochondria during apoptosis
of Drosophila cells (Dorstyn et al., 2002). Although an apoptosome-like complex
containing DRONC can form in vitro, the actual composition of this complex and
its role in caspase activation remains unknown (Dorstyn et al., 2002).

5.4

Other mechanisms

In vitro immunodepletion studies have shown that activated initiator caspases
(caspase-8 and -9 in particular) are necessary for the activation of effector caspases
(Slee et al., 1999). Although many effector caspases, including caspases-3, -6, and—
7, are capable of undergoing self-activation in vitro and when expressed in E.coli
(reviewed in Kumar and Lavin, 1996; Kumar, 1999), there is currently no evidence
for autoactivation under physiologic conditions. Among the apoptotic caspases,
caspases-2 is an unusual caspase, and its mechanism of activation is still a matter of
some debate (Kumar, 1999). Caspase-2 contains a CARD that has been shown to
homodimerize (Butt et al., 1998) and to interact with a CARD in the putative adapter
RAIDD (Duan and Dixit, 1997; Shearwin-Whyatt et al., 2000). However, definitive
evidence for a function of RAIDD in caspase-2 activation is still lacking (Shearwin-
Whyatt et al., 2000). Interestingly, in vitro cleavage and immunodepletion studies
suggest that caspase-3 mediates caspase-2 activation (Harvey et al., 1996; Slee et al.,
1999), and in the thymocytes derived from Apaf-1 and caspase-9 k/o mice, caspase-2
processing is severely impaired (O’Reilly et al., 2002), suggesting that caspase-2 may
be a downstream effector caspase. As caspase-2 is rapidly activated during apoptosis
induced by a variety of different signals (Harvey et al., 1997), and it has been shown
to act upstream of mitochondria to cause the release of cytochrome c and DIABLO
(Guo et al., 2002), it is possible that while caspase-2 can be activated by caspase-3-
mediated processing in the downstream caspase amplification loop, its primary role
may be as an initiator caspase.

6.

Regulation of caspase activation

As caspase activation is a key step in the initiation of apoptosis, it is tightly controlled
at various steps by a variety of proteins, including FLIP, the Bcl-2 family of proteins,
and the inhibitor of apoptosis proteins (IAP). Although low levels of caspase zymogens
are present in most cells, transcriptional regulation of caspases, particularly during
developmentally programmed cell death, is likely to play an important role.
Furthermore, caspase activation may also be regulated by compartmentalization, as
some caspases, such as caspase-2 and -12, are localized to specific cellular
compartments (Colussi et al., 1998a; Nakagawa et al., 2000).

40 GENETICS OF APOPTOSIS