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VOL 66 (4) 2000 IN VITRO INDUCTION OF APOPTOSIS BY CYCLOSPORINE A

significantly increased apoptosis was found after 4 hours at
concentrations of 10 µM. The difference in our data to those of other
investigators might be explained by the different CsA concentrations used
and differences in the treatment periods. CsA can serve as a specific
inhibitor at low concentrations and after short-term exposure, but not after
longer treatment with high concentrations.

CsA-induced MPT can have several consequences. One
consequence is the release of apoptosis-inducing factors (AIF), which
could be the cause of the increase in phosphatidylserine observed in the
outer leaflet of the cytoplasmic membrane, possibly by decreasing
flippase activities or inhibiting inactivated phosphatidylserine
translocases. Among others, cytochrome c may serve by itself as an AIF,
which, once it has been released from the mitochondria, can activate the
caspase cascade. Caspases are important effector molecules, which trigger
the biochemical events in apoptosis and ultimately execute apoptotic cell
death. The caspases are probably the most important effector molecules in
the apoptotic process. In general, caspases are present as inactive
proenzymes that are activated by specific proteases, in some cases by
autocatalysis (Guerrero and Arias 1998). Caspases are organized in
enzymatic cascades so that several upstream caspases are able to activate
downstream caspases. The presence of a protease cascade has been
suggested by the sequential appearance of proteolytic activity cleaving
fluorescent YVAD and DEVD substrates (Enari et al. 1996). In our study,
the participation of caspase-3 in the induction of CsA-induced apoptosis
can, at this time, be concluded only indirectly. In our experiments, we
observed a dose-dependent increase in caspase-3 activity after an
incubation of 20 hours with CsA, but not after 4 hours, although apoptosis
has also been clearly shown at this time point. This result principally
demonstrates that caspase-3 might be involved in the induction of CsA-
induced apoptosis after 20 hours. In comparison to this, the specific
caspase-3 blocker, Ac-DEVD-CHO was able to inhibit CsA-induced
chromatin condensation and fragmentation already after 4 hours, which
indicates that caspase-3 might be involved in the early apoptotic steps. As
well as the possible time-specific action of caspase-3, it also seems
possible that the activity of caspase-3 is not visible or is hidden after

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