(Received for publication, July 18, 1995; and in revised form, September 6, 1995)
From the
Bcl-2 and Bax are homologous proteins which can heterodimerize
with each other. These proteins have opposing effects on cell survival
when overexpressed in cells, with Bcl-2 blocking and Bax promoting
apoptosis. Here we demonstrate that gene transfer-mediated elevations
in Bcl-2 protein levels result in a marked increase in the steady-state
levels of endogenous p21 protein as determined by
immunoblotting in the Jurkat T-cell and 697 pre-B-cell leukemia cell
lines, but not in several other cell lines including CEM T-cell
leukemia, 32D.3 myeloid progenitor, PC12 pheochromocytoma, and NIH-3T3
fibroblasts. Steady-state levels of p21
protein were also
elevated in the lymph nodes of Bcl-2 transgenic mice in which a BCL-2 transgene is expressed at high levels in B-cells.
Northern blot analysis of BCL-2-transfected and
control-transfected Jurkat and 697 leukemia cells revealed no
Bcl-2-induced alterations in the steady-state levels of BAX mRNAs. In contrast, L-[
S]methionine pulse-chase analysis
indicated a marked increase in the half-life (t
)
of the p21
protein in BCL-2-transfected 697
cells compared to control-transfected cells (t
>24 h versus
4 h), whereas the rate of Bax
degradation was unaltered in Bcl-2-transfected CEM cells. The results
demonstrate that levels of the proapoptotic p21
protein
can be post-translationally regulated by Bcl-2, probably in a
tissue-specific fashion, and suggest the existence of a feedback
mechanism that may help to maintain the ratio of Bcl-2 to Bax protein
in physiologically appropriate ranges.
Among the more prominent regulators of Programmed cell death and apoptosis are Bcl-2 and its homologs. Gene transfer studies have shown that elevated levels of Bcl-2 protein can block or delay apoptotic cell death induced by a wide variety of stimuli and insults, suggesting that Bcl-2 controls a distal step in what may represent a evolutionarily conserved, final common pathway for cell death (reviewed in (1) ). The biochemical details of how the 25-26-kDa Bcl-2 protein controls cell life and death remain enigmatic, due at least in part to its lack of amino acid sequence homology with other proteins whose mechanism of action is known.
The Bcl-2 protein physically
interacts with several other proteins, including several homologous
proteins that constitute the Bcl-2 protein
family(2, 3, 4) . Some of the members of this
protein family are blockers of cell death akin to Bcl-2, whereas others
promote apoptosis and antagonize the function of Bcl-2 (reviewed in (1) ). The Bax protein shares 20% amino acid identity with
Bcl-2 and accelerates apoptosis when overexpressed by gene transfer
methods(3) . Bax forms heterodimers (or oligomers) with Bcl-2,
and mutagenesis studies have demonstrated that the ability of Bcl-2 to
heterodimerize with Bax is critical for the function of Bcl-2 as a
suppressor of cell death(5) . Immunohistochemical studies have
demonstrated that the BAX gene is widely expressed in
vivo, but is subject to tissue-specific and differentiation
stage-dependent regulation, which sometimes but not always correlates
with the apoptotic tendencies of cells in vivo(6) .
Expression of BAX is induced in some types of tumor cells by
radiation and chemotherapeutic drugs, in a p53-dependent manner,
suggesting an important role for the Bax protein in apoptosis induced
by genotoxic stress(7, 8, 9) . Furthermore,
reductions in Bax protein levels have been observed in
35% of
adenocarcinomas of the breast and correlate with poor responses to
chemotherapy in some subgroups of patients(10) . In addition,
rapid and striking elevations in p21
protein levels have
been described in ischemia-damaged neurons, implying a role for Bax in
at least some types of neuronal cell death that occur in the setting of
stroke (11) .
Taken together, these observations suggest
that BAX plays an important role in the control of cell death
in several clinically relevant contexts and suggest a need for a
greater understanding of the mechanisms that regulate the expression of
this gene. At present, however, relatively little is known about the
mechanisms that control the levels of the Bax protein. Clearly,
transcriptional mechanisms can play an important role, given that the BAX gene promoter contains several p53-binding sites and can
be strongly trans-activated by p53, at least in some types of
cells(9) . Here, we demonstrate that the levels of Bax protein
can also be controlled by post-translational mechanisms, with the Bcl-2
protein reducing the rate of p21 protein degradation in
some but not all types of cells.
Immunoblotting was performed as described in detail previously(6, 17) . Briefly, cells were lysed in 1% Triton X-100 solution as above, normalized for total protein content, and subjected to SDS-PAGE/immunoblot assay using either 0.1% (v/v) or 0.05% (v/v) rabbit anti-mouse Bax or anti-human Bax antiserum, respectively. Antibodies were detected using either biotinylated goat anti-rabbit Ig and an avidin-biotin-complex (ABC) reagent (Vector Laboratories, Inc.) containing horseradish peroxidase, followed by Vector SG-substrate or 3,3`-diaminobenzidine for colorimetric detection, or using horseradish peroxidase-conjugated goat anti-rabbit Ig and an ECL kit from Amersham, Inc. for autoradiographic detection.
We noticed that stable transfectants of the Jurkat human
T-cell leukemia cell line which overexpress Bcl-2 contained markedly
higher steady-state levels of endogenous p21 protein
compared to control transfectants (``Neo''). Fig. 1shows a representative example of immunoblot results
obtained for Jurkat-Bcl-2 and Jurkat-Neo cells. For this experiment,
detergent lysates were prepared from Jurkat-Bcl-2 and Jurkat-Neo cells,
normalized for total protein content (50 µg/lane), and subjected to
SDS-PAGE/immunoblot assay using an anti-human Bax
antiserum(10) . As shown, a strong 21-kDa band was seen in the BCL-2 transfectants, whereas only a very faint 21-kDa band was
observed in the Neo control transfectants (
50-fold less). In
contrast to Jurkat cells which contained very low levels of
p21
protein prior to transfection with BCL-2 expression plasmids, the basal steady-state levels of
p21
protein were much higher in another T-cell leukemia
line, CEM, and transfection with BCL-2 did not produce a
further elevation in Bax protein levels (Fig. 1). The blot shown
in Fig. 1was then reincubated with an anti-Bcl-2 antibody,
confirming the presence of
10-fold elevations in the relative
levels of p26-Bcl-2 protein in the BCL-2 transfectants of
Jurkat and CEM relative to the Neo control transfectants.
Figure 1:
Immunoblot analysis of Bcl-2 and Neo
transfectants of Jurkat and CEM leukemia lines reveals differential
regulation of p21 levels. Detergent lysates were prepared
from Bcl-2 (B) and Neo (N) transfectants of Jurkat
and CEM cells, normalized for total protein content (50 µg/lane),
and subjected to SDS-PAGE/immunoblot assay using anti-human Bax
antiserum followed by biotinylated secondary antibody, ABC-horseradish
peroxidase reagent, and 3,3`-diaminobenzidine for color detection
(brown), which revealed a 21-kDa band corresponding to the human Bax
protein (closed arrowhead). The same blot was then incubated
with anti-human Bcl-2 antiserum(24) , and colorimetric
detection was achieved similarly, except that Vector SG substrate was
employed (black), revealing the presence of the expected 26-kDa band
corresponding to human Bcl-2 (open arrowhead). (The original
brown (Bax) and black (Bcl-2) colors are not retained in the black and
white photograph shown.) Staining of the top of the blot (>50 kDa)
with Ponceau S confirmed loading of equivalent amounts of protein
samples for the pairs of BCL-2 and Neo transfectants (not
shown). In addition, a faint spurious band of
40-45 kDa can
be seen at equal intensity in Jurkat-Bcl-2 and Jurkat-Neo cells, thus
providing further confirmation that equivalent amounts of protein
samples were loaded.
This
analysis of steady-state p21 protein levels was then
extended to additional pairs of BCL-2- and Neo-transfected
cell lines, using immunoblot assays and antibodies specific for either
the human or mouse Bax proteins. As shown in Fig. 2, BCL-2 transfection resulted in increases in the steady-state levels of
p21
only in the Jurkat T-cell and 697 pre-B-cell leukemia
cell lines. In contrast, the relative steady-state levels of
p21
in BCL-2-transfected PC12, NIH-3T3, 32D.3,
and CEM cells were not detectably different from their Neo control
transfectant counterparts. Incubation of these blots with control
antibodies such as anti-F
-
-ATPase or anti-actin
confirmed the loading of equivalent amounts of total protein for the
pairs of samples (not shown). These results therefore suggest that the
influence of Bcl-2 on p21
protein levels is cell
type-specific.
Figure 2:
Immunoblot analysis of p21 levels in several pairs of Bcl-2- and Neo-transfected cell lines.
Detergent lysates were prepared from pairs of Bcl-2 and Neo
transfectants of human, rat, and mouse cell lines, normalized for total
protein content, and subjected to SDS-PAGE/immunoblot analysis using
either anti-mouse Bax (mouse and rat samples) or anti-human Bax
antiserum. Antibody detection was accomplished either by a colorimetric
method using Vector SG substrate (NIH-3T3, 32D.3, PC12, LN, Jurkat) or
by an ECL method (697, CEM). LN refers to detergent lysates
prepared from lymph nodes derived from either Bcl-2 transgenic (TG) or control nontransgenic littermate (C).
To explore the in vivo relevance of the
observation that Bcl-2 can produce elevations in the steady-state
levels of p21 in some cell lines, we compared the
relative levels of Bax protein in lymph nodes derived from Bcl-2/IgH
transgenic and nontransgenic littermate control mice(18) . Fig. 2shows a representative experiment demonstrating markedly
elevated levels of p21
protein in Bcl-2/Ig transgenic
lymph nodes compared to the nontransgenic littermate control. Similar
results were obtained in 3 of 3 transgenic/nontransgenic pairs of
age-matched animals (not shown).
Northern blot analysis was
performed to explore the effects of BCL-2 transfection on the
steady-state levels of BAX mRNA in Jurkat and 697 cells, where
gene transfer-mediated elevations in Bcl-2 resulted in substantial
increases in p21. As shown in Fig. 3(left
panel), the relative levels of the major
1.0-kbp as well as
the minor
1.5-kbp BAX mRNAs were not higher in the BCL-2 transfectants compared with the Neo control
transfectants. Reprobing the same RNA blot using a
[
P]glyceraldehyde phosphate dehydrogenase cDNA
confirmed loading of equivalent amounts of total RNA for all samples (Fig. 3, right panel). Bcl-2, therefore, does not cause
increases in p21
protein levels by stimulating elevations
in BAX mRNAs.
Figure 3:
RNA blot analysis of Bcl-2 and Neo
transfectants of Jurkat and 697 leukemia cells. Total cellular RNA (20
µg/lane) was subjected to Northern blot analysis using a P-labeled human BAX cDNA probe (left
panel). The arrowhead indicates the major 1.0-kbp BAX mRNA species. A minor
1.5-kbp BAX mRNA is also seen,
consistent with previous reports (3) . The same blot was
stripped and rehybridized with a human
[
P]glyceraldehyde phosphate dehydrogenase (GAPDH) probe (right panel) to verify loading of
equivalent amounts of RNA in all lanes.
In the absence of alterations in the relative
levels of BAX mRNA, it seemed likely that Bcl-2 causes
elevations in p21 protein levels by either increasing the
rate of BAX mRNA translation or by decreasing the rate of
p21
degradation. To explore this possibility, 697-Bcl-2
and 697-Neo cells were metabolically labeled with L-[
S]methionine for 2 h, and
p21
protein was immunoprecipitated immediately (0 h) or
at various times after chasing with unlabeled L-methionine. As
shown in Fig. 4A, the relative rates of
[
S]methionine incorporation into p21
were not reduced in 697-Neo cells relative to 697-Bcl-2 cells,
based on analysis of the Bax protein at 0 h before beginning the chase
with unlabeled L-methionine. Thus, the rate of synthesis of
p21
protein was not appreciably different for 697-Neo and
697-Bcl-2 cells. In contrast to 0 h, at 6 h after chasing out L-[
S]methionine,
S-Bax
protein levels were substantially reduced in 697-Neo cells but not in
697-Bcl-2 cells (Fig. 4A). By 12 h,
S-Bax
had almost disappeared from 697-Neo cells and by 24 h no
S-Bax was detected, whereas
S-Bax protein
levels were nearly constant for the first 24 h after chasing out L-[
S]methionine in the 697-Bcl-2 cells (t
4 h for Neo versus >24 h
for Bcl-2). In parallel with the disappearance of the 21-kDa Bax band
in 697-Neo cells upon chasing with unlabeled methionine, a new species
of
17 to 18 kDa appeared, suggesting a possible precursor-product
relation. Immunoprecipitations performed using normal rabbit serum(N)
instead of anti-Bax antiserum or using anti-Bax antiserum that had been
preadsorbed with Bax peptide (P) confirmed the specificity of these
results, in that no 21-kDa band corresponding to Bax was detected. An
additional 26-kDa band was also co-immunoprecipitated with p21
in 697-Bcl-2 but not 697-Neo lysates (Fig. 4A).
This 26-kDa band represents Bcl-2 in physical association with
p21
, based on experiments where the Bax
immunoprecipitates were analyzed by immunoblotting using Bcl-2-specific
antibodies (data not presented). (Note that a slightly faster migrating
protein at the
25-kDa band is also seen in 697-Neo cells which
appears to be nonspecific, since it was also present when normal rabbit
serum was employed instead of anti-Bax antiserum. This nonspecific
protein is also seen to a lesser extent in 697-Bcl-2 cells just below
the 26-kDa band corresponding to Bcl-2.) Similar results were obtained
for Jurkat-Neo and Jurkat-Bcl-2 cells, except that the rate of
degradation of Bax in Jurkat-Neo cells was faster than in 697 cells,
precluding an accurate estimate of the half-life of Bax protein under
the experimental conditions employed here (not shown).
Figure 4:
[S]Methionine
pulse-chase analysis of p21
degradation rates in Bcl-2
and Neo transfectants of 697 and CEM leukemia line. In A,
Bcl-2 and Neo transfectants of 697 cells were metabolically labeled
with L-[
S]methionine and either lysed
immediately (0 h) or cultured for 6-24 h in medium containing
unlabeled methionine prior to preparing detergent lysates and
performing immunoprecipitations using anti-human Bax antiserum. In some
cases, either preimmune serum (N) was used for
immunoprecipitations or the anti-Bax antibody was preadsorbed with 10
µg of Bax peptide (P) as controls for verifying
specificity of the antibodies. In the Bax immune complexes prepared
from 697-Bcl-2 cells, an additional 26-kDa band was seen which probably
represents Bcl-2 protein. In B, Bcl-2 and Neo transfectants of
697 and CEM cells were labeled with L-[
S]methionine, and immunoprecipitates
were prepared using anti-human Bax antibody immediately (0 h) or after
4 h of culture in medium containing unlabeled methionine. Gels were
analyzed by autoradiography (top), and p21
bands
were quantified using a
-scanner, with the results expressed
relative to the 0-h value for each stable transfectant. Data are
representative of two experiments.
In addition,
the effects of Bcl-2 transfection on p21 protein
stability were compared in 697 cells with CEM cells. Since Bcl-2 did
not cause elevations in p21
in CEM cells, we would not
expect the half-life of p21
to be prolonged in these
leukemic cells, unlike in 697 cells where Bcl-2 did increase the
relative levels of p21
. Again, a
[
S]methionine pulse-chase analysis was
performed, comparing the relative amounts of
S-Bax at 0 h
before chasing L-[
S]methionine and at 4
h after chase, which was chosen for these comparisons because it
represents the approximate t
of Bax in 697-Neo
cells. As shown in Fig. 4B, the relative levels of
S-Bax remained essentially unchanged in CEM-Neo and
CEM-Bcl-2 cells at 4 h after chasing with unlabeled L-methionine, suggesting that the t
of
Bax is comparatively long in these cells irregardless of their levels
of Bcl-2 protein. In contrast, relative levels of
S-Bax
were reduced by approximately one-half at 4 h after chasing with
unlabeled [
S]methionine in 697-Neo cells but not
in 697-Bcl-2 cells.
The data presented here provide the first evidence that
levels of a Bcl-2 family protein, in this case p21, can
be regulated through post-translational mechanisms. Gene
transfer-mediated elevations in Bcl-2 protein resulted in increases in
p21
by decreasing the rate of Bax protein degradation in
some types of cell lines. A reasonable speculation therefore is that
the interaction of Bcl-2 with p21
, leading to formation
of heterodimers or complexes with other stoichiometry, somehow
stabilizes the Bax protein in some types of cells, thus leading to
increases in the steady-state levels p21
protein. We are
currently attempting to express a variety of Bcl-2 mutants in Jurkat
T-cells that either do or do not retain the ability to bind to Bax as
an approach to testing this hypothesis directly. Consistent with this
notion, a 26-kDa protein representing Bcl-2 co-immunoprecipitated with
Bax in 697-Bcl-2 cells where p21
had a long half-life (t
> 24 h) but not in 697-Neo cells where
p21
turnover was considerably more rapid (t
4 h).
It remains to be determined
what the explanation is for the cell type specificity observed, where
Bcl-2-mediated up-regulation of p21 protein levels in
some cell lines but not others. Interestingly, even closely related
cells such as the human T-cell acute lymphocytic leukemia lines Jurkat
and CEM exhibited markedly different responses to Bcl-2 in terms of
effects on p21
protein levels. Despite the fact that
Jurkat and CEM are both T-cell acute lymphocytic leukemias, however,
they possess immunophenotypes consistent with different stages of
thymic development, i.e. CD4
CD8
(mature thymocytes; Jurkat) and
CD4
CD8
(immature thymocytes; CEM).
Presumably, whatever factors that contribute to rapid degradation of
Bax in cell lines such as 697 and Jurkat are not operative in CEM and
other cell lines where Bcl-2 did not induce increases in Bax protein
levels. These factors could include differences in the levels or
activity of (i) proteases, (ii) kinases or phosphatases that
theoretically could control phosphorylation of p21
, or
(iii) non-Bcl-2 family proteins that might bind to p21
.
Of particular note with regard to mechanisms of protein degradation is
the observation that several recently described Bcl-2 binding proteins
contain either PEST sequences (Nip-1, Nip-2, Nip-3) or ubiquitin-like
domains (BAG-1) which conceivably might target protein complexes that
contain these Bcl-2 binding proteins for proteolytic
degradation(19, 20) . Moreover, the Bcl-2 homologous
protein Mcl-1 contains PEST sequences and has been shown to bind to
p21
(4, 21) . Alternatively, it could be
that in addition to Bcl-2, other antiapoptotic members of the Bcl-2
protein family such as Bcl-X
or Mcl-1, which can also
heterodimerize with Bax(2, 4) , are constitutively
present at higher levels in cell lines such as CEM and these proteins
perhaps may similarly stabilize p21
in a manner similar
to Bcl-2. In this case, the supposition would be that these other
Bcl-2-like proteins are already present at optimal levels for
suppressing p21
degradation and that gene
transfer-mediated increases in Bcl-2 protein therefore provide no
further benefit. Immunoblot experiments not shown here indicate that
all of the cell lines tested here express Bcl-X
, Mcl-1, or
both, although no correlations with Bax levels were apparent.
Nevertheless, the more rapid degradation of Bax may be to some extent
analogous to the rapid degradation of unpaired subunits of
multipolypeptide complexes(22, 23) .
Gene transfer
studies in mammalian cells have shown that elevations in Bcl-2 protein
increase resistance of cells to various apoptotic stimuli, whereas
increases in Bax can accelerate cell death in the setting of growth
factor deprivation. Presumably, therefore, the ratio of Bcl-2 to Bax
controls the relative sensitivity or resistance of cells to apoptosis.
One prediction of the finding that Bcl-2 can induce elevations in
p21, therefore, is that it may represent a feedback
mechanism for maintaining Bcl-2/Bax ratios within a physiologically
appropriate range. Since experimental manipulations that increase Bcl-2
relative to Bax have been shown to prolong cell survival and block
apoptosis induced by many insults, the positive effects of Bcl-2 on
p21
half-life may help to limit cell longevity and thus
reduce the risk of tumorigenesis. Alternatively, it has not been
excluded that Bcl-2/Bax heterodimers represent a molecular entity that
promotes cell survival, not unlike the
- and
-chains of some
cell surface receptors such as integrins and T-cell antigen receptors.
In this case, Bcl-2-mediated up-regulation of p21
would
constitute a positive feedback loop. Thus, until the functional
properties of Bax/Bax homodimers, Bax/Bcl-2 heterodimers, and
Bcl-2/Bcl-2 homodimers are better understood in terms of their
biochemical influence on cell death pathways, it is difficult to
predict the physiological consequences of the Bcl-2-mediated
stabilization of the p21
protein described here.
Nevertheless, these findings indicate that it may be important to
determine the effects of Bcl-2 on Bax protein levels before
interpreting the biological significance of experiments where gene
transfer-mediated elevations in Bcl-2 are employed to assess the
relative Bcl-2 dependence or independence of various apoptotic stimuli
and pathways.