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is thought to cause decrease of the mitochondrial membrane potential (Dc)
(Zamzami et al., 1995, 1996; Zoratti and Szab
Á
, 1995), swelling of the organ-
elle, rupture of the outer membrane, and release of cyt c ( Liu et al., 1996) and/
or AIF (Susin et al., 1996, 1999) from the intermembrane space. Both the latter
compounds are able to induce nuclear apoptosis, i.e., apoptotic changes in iso-
lated nuclei, that can be inhibited by Bcl-2 protein activity (Susin et al., 1996)
and by protease inhibitors (Marchetti et al., 1996). AIF seems to activate
apoptosis directly without the activation of caspases (reviewed in ( Daugas et al.,
2000)), whereas cyt c activates caspases through its e¨ects on Apaf-1 cytosolic
factor (Zhou et al., 1997). Upon binding of cyt c, Apaf-1 becomes competent
at binding and activates procaspase-9 ( Li et al., 1997; Zhou et al., 1997). The
antiapoptotic or proapoptotic e¨ect of Bcl-2 family members may reside in the
regulation of the release of such apoptogenic molecules into cytosol. However,
the precise mechanism by which they promote or prevent cell death is far from
clear.
On the whole, it is evident that mitochondria are deeply involved in cell
death, and it has been proposed that they might act as the central executioner of
apoptosis (Susin et al., 1997). It has also been suggested that the fall in Dc
could be a common feature of the apoptotic program. Several groups inves-
tigating this point have produced con¯icting (or contradictory) results, but
these were likely due, at least in part, to di¨erent experimental models, or to the
interpretation of data obtained using di¨erent dyes. In fact, Dc has been mea-
sured by ¯ow cytometry or confocal microscopy using a variety of ¯uorescent
probes with di¨erent sensitivities to this parameter ( Lopez-Mediavilla et al.,
1989; Salvioli et al., 1997). In any case, it has been demonstrated that the re-
lease of cyt c can occur without a simultaneous drop in Dc ( Kluck et al., 1997;
Yang et al., 1997). It is not clear how a protein like cyt c can transit from the
intermembrane space into the cytosol without disruption of Dc. It has been
proposed that pores are created ex novo in the outer membrane, allowing some
cyt c to escape, while leaving the permeability of the inner membrane intact
(Vander Heiden, et al., 1997). Therefore, it has been postulated that the release
of apoptogenic molecules, and not the collapse of Dc, could be the true link
between mitochondria and apoptosis ( Lemasters et al., 1998). On the other
hand, signi®cant portions of the Bcl-2 protein are also integrated either into the
membranes of the endoplasmic reticulum (ER) or into the nuclear envelope.
ER-targeted Bcl-2 retained the ability to block apoptosis in some experimental
models (Zhu et al., 1996), suggesting that Bcl-2 could possess some non-
mitochondrial, still-unknown mechanisms for promoting cell survival. In this
perspective, the ®nding that some ¯uorescent probes whose accumulation oc-
curs mostly on ER membranes rapidly lose ¯uorescence intensity during early
phases of apoptosis may suggest that these organelles, and not only mitochon-
dria, are precociously involved in cell-death regulation. Finally, it has been
shown that none of the following events represents a point of no return in the
apoptotic process: loss of DC, changes in mitochondrial permeability transition
(MPT ), or mitochondrial swelling (Minamikawa et al., 1999), taking into ac-
