Genetics of Apoptosis

(Barry) #1

as shown in Figure 1. Chromatin degradation to nucleosome-sized fragments
(laddering) was also observed (Cikala et al., 1999). All these features of the cell-death
phenotype in Hydra are indistinguishable from the apoptotic phenotype in higher
animals and suggest that the mechanism of apoptosis is the same in Hydra and higher
animals.
The use of fluorogenic substrates specific for caspase-3 (DEVD-AMC) has
demonstrated a dramatic increase in caspase activity in colchicine-treated animals.
Moreover, two cDNAs have been cloned from Hydra with homology to caspase-3
from higher animals. They encode typical procaspase sequences with an amino
terminal prodomain followed by large and small subunits (Cikala et al., 1999). More
recent experiments have shown that Hydra caspase-3A, when expressed in bacteria,
undergoes autocatalytic cleavage between the large and small subunit, leaving the
prodomain attached to the large subunit. Cleavage occurred at the C-terminal to the
sequence IRKD. This is clearly a caspase cleavage site, although the sequence is
somewhat atypical. In accordance with this unusual substrate specificity, active site
labeling could be achieved only by using the non-specific caspase inhibitors, zEK
(biotin)D-AOMC and z-VAD-AMC. None of the commercially available specific
caspase inhibitors, such as the caspase-3 inhibitor DEVD-FMK or the caspase-1
inhibitor YVAD-FMK, could compete with the nonspecific inhibitors (Böttger and
David, unpublished observations).


Figure 1. Single epithelial cell of Hydra containing three vacuoles, each with an apoptotic cell.


Hydra specimens were treated with wortmannin to induce apoptosis. Epithelial cells acting as
nonprofessional phagocytes rapidly engulfed the apoptotic cells. (a) Phase-contrast micrograph
of an e p it he l ial c el l f r o m a t r e at e d p ol yp. ( b ) DA P I f l uo re sc e nc e o f t h e sam e c e ll sh owi ng n or m a l
nuclear morphology of the epithelial cell and pyknotic condensation in nuclei of the three
apoptotic cells. Scale bar: 10 μm.


EVOLUTION OF CELL DEATH 153
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