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ARMIN WOLF ANAL REAL ACAD. FARM.

4 hours in the case of CsA. Under the same conditions, the cyclosporin
derivative, IMM 125, an inducer of apoptosis which is twice as strong as
CsA, significantly increased caspase-3 activity after 4 hours (paper in
preparation). The differences in the CsA-induced, caspase-3 activity dose-
response curve and the chromatin condensation/fragmentation dose-
response curves might be due to analytical reasons. While the natural
substrate of caspase-3 is poly-ADP-ribose-polymerase (PARP)
(Thornberry and Lazebnik 1998, Inayat-Hussain et al. 1997), in our
analytical test system we used an artificial substrate, Ac-DEVD modified
7-amino-4-methyl coumarin (AMC), which could have a different affinity
to the enzyme, and thus be less sensitive. In addition, the strong
background noise signal in the analytical assay of caspase-3 activity did
not allow the sensitive detection of very small increases in comparison
with controls.

Caspase-6 activity was also found to be statistically significantly
increased by CsA, but these effects were only minor. Its minor role was
also demonstrated by the relatively weak effect of the specific inhibitor,
which showed an inhibitory effect on chromatin condensation and
fragmentation only after 20 hours at a low magnitude of inhibition.

The results obtained by Ac-DEVD-CHO suggest that necrosis
might be in close relation to apoptosis. In our experiments, we also found
that the caspase-3 inhibitor not only prevented CsA-induced apoptosis,
but also LDH release. Since LDH leakage indicates plasma membrane
damage, this is a parameter which, under the given conditions, could
serve to determine necrosis. Results obtained by TEM would support such
an assumption, especially under in vitro conditions. Some apoptotic cells
with necrotic properties were seen. Within tissue, apoptotic bodies usually
undergo rapid phagocytosis. However, in cell cultures, such bodies can
undergo spontaneous degeneration with swelling and membrane rupture
(Eanshaw 1995). The necrosis observed in the cultures in the present
study could partly be “secondary necrosis” of apoptotic bodies (Eanshaw
1995). It seems possible that transition from apoptosis into necrosis may
also occur in vivo.

Our results suggest that the mechanism of CsA-induced apoptosis
proceeds from disruption of mitochondrial membrane potential to

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