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

destruction of the mitochondrial membrane, resulting in cytochrome c
release into the cytosol. This results in an activation of caspases,
especially caspase-3, and, to a less extent, caspase-6. As a consequence,
DNA is attacked, as observed by condensation and fragmentation of
chromatin.

Although Ac-DEVD-CHO and Ac-VEID-CHO inhibited CsA-
induced chromatin condensation and fragmentation, the blockage of
apoptosis was, in all cases, not complete. In our experiment, we used
inhibitor concentrations which were supposed to be specific according to
the current literature (Inayat-Hussain et al. 1997). Thus, it can be
assumed, that the concentrations to achieve full inhibitory action were
sufficient to demonstrate the involvement of both of these enzymes. That
we did not succeed in total inhibition may indicate that not only caspases
are involved in the process of CsA-induced apoptosis, but also other
mechanisms.

We cannot exclude that there exist other mechanisms, parallel
with the activation of caspases, which result in CsA-induced apoptosis. It
is also possible that intracellular calcium plays a role in this complex
mechanism. This would be consistent with the hypothesis of Jewell et al.
(Jewell et al. 1982), who postulated that morphological changes in the
plasma membrane are associated with disturbances in intracellular
calcium homeostasis. It is known that an increase in intracellular calcium
concentration may directly cause toxicity. Free calcium can serve as a
mediator of cytotoxicity by means of activating catabolic pathways by
which important cellular macromolecules such as proteins, lipids and
nucleic acids are degraded (Orrenius 1993, Richter and Kass 1991).
Activation of Ca2+-dependent endonucleases could lead to the typical
apoptotic DNA ladder pattern (Guerrero and Arias 1998). Although
inhibition of CsA-mediated Ca2+ efflux from mitochondria has been
described by Richter et al. and several other authors (Richter and Kass
1991, Nicchitta et al. 1985, Kehrer et al. 1993), there is good evidence
that the uptake of extracellular Ca2+ is enhanced by CsA in intact
hepatocytes (Ellouk-Achard et al. 1997), in other cell types, and also in
liposomal fractions (Wolf et al. 1997, Meyer-Lehnert and Schrier 1989,

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