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

supported by ultrastructural evaluations using TEM. TEM was used for
qualitative assessment of the structural changes and did not permit a full
quantitative assessment of the findings. With TEM it was possible to
demonstrate that CsA induced apoptosis according to classical criteria and
to distinguish the effects observed from necrosis. Apoptosis appeared as
typical cell shrinkage accompanied by condensed cytoplasm with focally
dilated endoplasmic reticulum, structurally intact mitochondria and other
organelles compacted together and maintaining their integrity, formation
of cytoplasmic protuberances on the cell surface (blebbing) and
cytoplasmic bodies, nuclear shrinkage with invagination of nuclear
membrane, and massive deposits of chromatin in compact masses
adjoining the nuclear envelope. It was also possible to clearly distinguish
necrosis characterized by swelling of all cytoplasmic compartments,
swelling and disappearance of mitochondrial cristae, disintegration of
membranes and accumulation of large dense granules in the
mitochondrial matrix, detachment of ribosomes from rough endoplasmic
reticulum membranes, and nuclear swelling with clumping of loosely
textured nuclear chromatin (Wyllie et al. 1980).

The results obtained from morphological investigations were
supported by the specific determination of DNA fragmentation using the
TUNEL method. The results showed that CsA specifically causes DNA
damage by the generation of 3’-OH end fragments. This method is
considered to be specific because of the binding of the terminal
deoxynucleotidyl transferase (TdT) to exposed 3’-OH ends of DNA
fragments generated in response to apoptotic stimuli (Gavrieli et al.
1992). The loss of plasma membrane asymmetry is another feature, and is
an early process in apoptosis, preceding apoptotic DNA degradation
(Eanshaw 1995, Martin et al. 1995).

In the present investigation, membrane phosphatidylserine
distribution was used as a sensitive parameter of apoptosis to indicate
plasma membrane asymmetry. In normal cells, phosphatidylserine is
located exclusively in the inner cytoplasmic surface of the membrane. In
apoptotic cells, phosphatidylserine flips within the cell membrane to
become located on both surfaces. This is an early event in the apoptotic

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