p53 protein. Changes in Bax expression have been reported in some pathologic
conditions. For example, in one-third of breast adenocarcinomas, Bax expression
levels have been found to be decreased (Krajewski et al., 1995a). For some tumors, a
low Bax expression has been correlated with a poor prognosis for the patients (Ito et
al., 1999). However, in most, if not all, cells, the multidomain proteins are present
in inactive forms.
In normal cells or tissues, Bax is predominantly localized in the cytosol as a
monomer (Hsu and Youle, 1998; Antonsson et al., 2000). Bax translocation from
the cytosol to the mitochondria after apoptotic stimulation has been demonstrated
in several systems. Deprivation of interleukin-3 from FL5.12 hematopoietic cells
induced Bax translocation (Goping et al., 1998; Gross et al., 1998). Translocation of
Bax has also been demonstrated after withdrawal of the cytokine interleukin-7 from
T cells (Khaled et al., 1999). In this study, an increase of intracellular pH was found
to coincide with Bax translocation. However, other studies have shown cytosolic
acidification during apoptosis. Using a pH-sensitive, green fluorescent protein,
Matsuyama et al. (2000) showed that mitochondrial dependent apoptosis resulted in
cytosolic acidification and mitochondrial alkalinization. The pH changes were not
detected in death receptor-triggered apoptosis. Whether the change in intracellular
pH is a causal or resultant event in mitochondrial dependent apoptosis remains
unclear.
Activation and translocation of Bax have been shown to be accompanied by
conformational changes in the protein, resulting in changes in its quaternary
structure. When Bax was activated through incubation with Bid, the conformation
of the N-terminal domain changed, making this domain reactive to antibodies that
do not react with the unactivated protein (Desagher et al., 1999). Changes in the C-
terminal domain have also been shown to occur after Bax activation (Nechushtan et
al., 1999). Upon activation, the C-terminal α-helix is removed from the BH3 cleft,
making the BH3 domain accessible to interactions leading to complex formation
(Suzuki et al., 2000). After activation, Bax is found inserted into the outer
mitochondrial membrane as large oligomers (Antonsson et al., 2001). Bcl-2 inhibits
Bax activation and oligomerization (Antonsson et al., 2001; Mikhailov et al., 2001).
A study by Mahajan et al. (1998) demonstrated direct interactions between
fluorescent-labeled Bax and Bcl-2 at the mitochondria by fluorescence resonance
energy transfer (FRET). However, Mikhailov et al. (2001) could not show any direct
interactions between Bax and Bcl-2 through immunoprecipitation or cross-linking
in Bcl-2-overexpressing cells where Bax oligomerization was prevented. Thus, the
molecular mechanism for Bcl-2 prevention of Bax activation remains unclear and
might depend on cell type or the apoptotic stimulation. Tumor necrosis factor (TNF)-
alpha-mediated cell death was recently shown to be associated with the formation of
Bax protein complexes of 500 kDa. Conformational changes in both the C—and N-
terminal domains were associated with complex formation. Furthermore, the
adenovirus E1B 19K protein, a Bcl-2 homolog, inhibited Bax oligomerization and
blocked TNF-alpha-induced cell death (Sundararajan and White, 2001).
MITOCHONDRIA IN APOPTOSIS INDUCTION 129