Genetics of Apoptosis

initiation site of the apoptotic wave accumulate Ca2+ very efficiently, perhaps because
they are in close apposition with ER Ca2+-release sites and are intrinsically specialized
for Ca2+ uptake, or because some mitochondria receive a greater degree of apoptotic
stress, which switches them into an excitable state and marks them as initiation sites
for wave propagation (Pacher and Hajnoczky, 2001). One might imagine that when
a cell receives an apoptotic stimulus it may signal to BH3-only molecules to prime
mitochondria to release Cyt.c and other IMS proteins. However, the coordinated
release of these molecules is not initiated until privileged mitochondria receive an ER
Ca2+ spike that causes a transient opening of the PTP and release of Cyt.c and matrix
Ca2+, initiating a second Ca2+ wave that is received and regenerated by neighboring
mitochondria. These mitochondria, in turn, propagate the wave, and as this cycle is
repeated over and over, a traveling wave of apoptotic mitochondrial transition spreads
through the cell, allowing a coordinated and complete release of Cyt.c and cellular
progression into the execution phase of apoptosis. This model reinforces the
prediction that mitochondrial apoptosis in intact cells proceeds through a ‘two-hit’
mechanism (Pinton et al., 2001) that requires a factor to operate directly on
mitochondria as well as a costimulatory signal, in this case from the ER, that
coordinates the release of Cyt.c between mitochondria (Figure 2 ).

3.5

Bcl-2 family members regulate ER Ca2+ homeostasis

Although the function of Bcl-2 and Bcl-xL is best characterized at the mitochondrion,
both proteins locate to other intracellular membranes such as the ER and nuclear
envelope. Numerous reports suggest that Bcl-2 can alter ER Ca2+ homeostasis;
however, there are discrepancies in how it does so. Several groups have reported that
Bcl-2 increases the ER Ca2+ content and/or prevents ER Ca2+ release during apoptosis
(Lam et al., 1994; Distelhorst et al., 1996; He et al., 1997; Ichimiya et al., 1998). In
contrast, using probes that specifically target the ER, two groupsfound that
overexpression of Bcl-2 reduces the steady-state level of [Ca2+]ER by increasing the
permeability of the ER membrane to Ca2+ in HeLa, mouse A20, and HEK-293 cells
(Foyouzi-Youssefi et al., 2000; Pinton et al., 2000). Consistent with this latter finding,
Pinton et al. (2001) found that manipulations that lead to decreased [Ca2+]ER protect
cells against apoptosis, whereas manipulations that increase [Ca2+]ER sensitize cells to
apopotosis. Thus, the primary role for Bcl-2 at the ER may be to lower the ER calcium
reservoir, which could dismantle IP3R/RyR-mediated signals during apoptosis. The
studies described above were carried out with wild-type Bcl-2, which locates to
nuclear, mitochondrial, and ER membranes; therefore, it cannot be ruled out that
the observed effects on ER Ca2+ stores are caused by its actions at other cellular
locations. Nonetheless, Bcl-2 can exert an antiapoptotic action at the ER, since Bcl-2
that is targeted exclusively to the ER by replacing its membrane insertion sequence
with that of cytochrome b5 (Bcl-2cb5) can inhibit apoptosis induced by ER stress
agents, cermide, Myc, ionizing radiation, or BAX overexpression, but not by
etoposide or death receptor signals (Hacki et al., 2000; Annis et al., 2001; Rudner et

112 GENETICS OF APOPTOSIS