Page 88 - 66_04

ARMIN WOLF ANAL REAL ACAD. FARM.

process, which is used to detect apoptotic cells before gross
morphological changes become visible. The phospholipid-binding protein
Annexin V has a high affinity for phosphatidylserine and serves as a very
specific marker for apoptotic cells. In the current study, CsA-induced
Annexin V reactive cells increased dosedependently, at all time points, in
parallel with chromatin changes and DNA degradation.

Annexin V staining, chromatin condensation and fragmentation
and the TUNEL assay have also a certain potential to give positive results
with necrotic cells (Gold et al. 1994, Vermes et al. 1995). By means of
ultrastructural investigation with TEM we could exclude the possibility of
necrosis at 4 hours after CsA treatment at all concentrations. Exclusion of
necrosis can also be supported by the determination of LDH activity in
the cell culture supernatant, proof of the integrity of the outer cell
membrane, a typical criteria for necrotic cell death. The results obtained
from the parameters of apoptosis applied after 4 hours can therefore be
considered to be highly specific for the induction of apoptosis.

There is no general mechanism for the induction of apoptosis;
induced by different mediators, it may occur by different mechanisms.
Mitochondria, however, are thought to play a central role in the activation
of apoptosis, induced by multiple agents, by a mechanism that involves
breakdown of mitochondrial membrane potential and release of
cytochrome c and its binding to a multiprotein complex that activates
caspase-3 (Pan et al. 1998).

Our data strongly suggest that CsA induces apoptosis by
decreasing mitochondrial membrane potential, which is followed by MPT
as determined by cytochrome c release from the mitochondria. This
finding is in conflict with the current literature. CsA has been described
by many authors as a very specific inhibitor of mitochondrial membrane
potential and permeability transition in isolated mitochondrial fractions
and in isolated hepatocytes (Lemasters et al. 1998), when incubated for 1
hour at concentrations in the nanomolar range. Under our experimental
conditions, we found a rapid loss of mitochondrial membrane potential 1
hour after CsA treatment at 1 µM. After 1 hour of incubation with 50 µM
CsA, mitochondrial membrane potential decreased to 50 % of that of
control cells. CsA-mediated MPT was observed after 4 hours, and

26