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INTRODUCTION |
The signaling pathways involved in apoptosis have been intensely
studied in the last few years due to their role in many disease states
including cancer and neurodegenerative disorders. One central player is
the Bcl-2 family of proteins, which can either promote cell survival
(Bcl-2, Bcl-XL, A1, Mcl-1, and Bcl-W) or promote cell death (Bax, Bak,
Bcl-XS, Bad, Bid, Bik, Bim, Hrk, Bok) (reviewed in Ref. 1). The
relative amounts or equilibrium between these pro- and antiapoptotic
proteins influence the susceptibility of cells to apoptosis. As such,
many cancer cells up-regulate the antiapoptotic members as a mechanism
to guard against programmed cell death. Although much work has been
carried out studying the protective role of Bcl-2 and Bcl-XL in
response to many different apoptotic stimuli, relatively little is
known about the other antiapoptotic family members or the specific role
of each isoform.
Endothelial cells play a pivotal role in modulating the inflammatory
response. Stimulation of endothelial cells by tumor necrosis factor
(TNF-
)1 activates the
transcription factor NF-
B, leading to induction of proinflammatory
genes. TNF-
is also known to trigger programmed cell death, which in
most cells is controlled by the simultaneous up-regulation of
cytoprotective genes (Refs. 2-4; reviewed in Ref. 5). Because this
anti-death activity requires de novo protein synthesis,
cells resistant to the cytotoxic effects of TNF-
, including
endothelial cells, can be rendered sensitive by pretreatment with
either RNA or protein synthesis inhibitors. To date, several
TNF-
-inducible cytoprotective genes have been identified and have
been shown through overexpression techniques to protect cells, at least
in part, from death induced by TNF-
and cycloheximide/actinomycin D. This growing family represents a diverse range of proteins and includes
manganous superoxide dismutase (6), plasminogen activator inhibitor
type 2 (7), the zinc-finger protein A20 (8), the
cyclin-dependent kinase inhibitor p21 (9), xiap, a member
of the IAP family (10), IEX-1L, a protein of unknown function (11), the
Bcl-2 family member A1 (12), and combinations of c-IAP-1, c-IAP-2,
Traf1, and TRAF2 (13).
We initiated studies aimed at addressing the role of A1 as a
TNF-
-inducible cytoprotective gene and comparing its function to
Bcl-XL, which is expressed constitutively in human umbilical vein
endothelial cells (HUVEC). In order to help address the importance of
each isoform in normal tissue, highly selective and potent antisense
inhibitors were utilized in this study. We show that inhibition of A1
alone does not render HUVEC susceptible to TNF-
-induced apoptosis.
However, inhibition of Bcl-XL caused HUVEC to undergo apoptosis and
sensitized cells to treatment with either ceramide or staurosporin.
Furthermore, Bcl-XL protected cells from both caspase-dependent and -independent mechanisms of
Ym disruption.
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MATERIALS AND METHODS |
Cells and Reagents--
HUVEC were obtained from Clonetics (San
Diego, CA) and cultivated in endothelial growth medium supplemented
with 10% fetal bovine serum. Cells were used between passages 2 and 5. Opti-MEM, Lipofectin reagent, and fetal bovine serum were purchased
from Life Technologies, Inc. TNF-
was obtained from R & D Research (Minneapolis, MN), Bcl-X antibody and the ribonuclease protection assay
were from Pharmingen (San Diego, CA). Caspase 3 antibody was from
Transduction Laboratories (Lexington, KY), and MitoTracker Orange
CMTMRos was purchased from Molecular Probes (Eugene, OR). SDS-polyacrylamide gels were obtained from NOVEX (San Diego, CA). Staurosporin, C6-ceramide,
benzyloxycarbonyl-Val-Ala-Asp(OMe)-CH2 (Z-VAD.fmk), and
carbonyl cyanide m-chlorophenylhydrazone were purchased from
Calbiochem. Hybond N+ nylon membranes and the ECL Plus detection kit
were from Amersham Pharmacia Biotech.
Oligonucleotide Synthesis and Sequences
Used--
2'-O-Methoxyethyl chimeric antisense
oligonucleotides were utilized for all experiments. The A1 antisense
oligonucleotide and its control contained phosphodiester linkages in
the 2'-O-methoxyethyl regions and phosphorothioate linkages
in the 2'-deoxynucleotide regions, which support RNase H activity in
cells (14). The Bcl-XL antisense oligonucleotide and its control
contained phosphorothioate linkages throughout. All oligonucleotides
were synthesized using an Applied Biosystems 380B automated DNA
synthesizer and purified as described previously (15, 16).
Oligonucleotide and TNF-
Treatment of HUVEC--
HUVEC were
treated with oligonucleotides at ~80% confluence (17, 18). Briefly,
cells were washed three times with prewarmed (37 °C) Opti-MEM.
Oligonucleotides were premixed with 10 µg/ml Lipofectin reagent in
Opti-MEM at the desired concentration and applied to washed cells.
HUVEC were incubated with the oligonucleotides for 4 h at
37 °C, after which the medium was removed and replaced with standard
growth medium. For experiments carried out with A1 antisense
oligonucleotide, the cells were allowed to recover 16-18 h after
oligonucleotide treatment, prior to induction with TNF-
(10 ng/ml),
C6-ceramide (5 µM), staurosporin (1 nM), or Z-VAD.fmk (100 µM). Because Bcl-XL is expressed
constitutively, treatment with TNF-
(10 ng/ml), C6-ceramide (5-10
µM), staurosporin (1-2 nM), or Z-VAD.fmk
(100 µM) was delayed 24 h after oligonucleotide treatment. This delay period allowed time for reduction in Bcl-XL protein prior to the addition of apoptotic stimuli.
Northern Analysis--
Total cellular RNA was isolated either
4 h after TNF-
induction (A1 antisense experiments), or 24-48
h after oligonucleotide treatment (Bcl-XL antisense experiments)
utilizing Quiagen RNeasy Kit. Isolated RNA was separated on a 1%
agarose/formaldehyde gel, transferred to a Hybond N+ nylon membrane
over-night, and UV-cross-linked (Stratalinker 2400, Stratagene). Blots
were hybridized several hours with random primed
32P-labeled probes (Bcl-XL) or single-stranded PCR
32P-labeled probes (A1) as described by Bednarczuk et
al. (19). Both A1 and Bcl-XL cDNA's were generated by reverse
transcription-polymerase chain reaction carried out with HUVEC total
RNA. Polymerase chain reaction was subsequently utilized to amplify
bases 22-684 of the human A1 gene and bases 34-856 of the human
Bcl-XL gene. The primer utilized to generate the single-stranded A1
probe was 5'-AGAAGTATGTGTTGGCAATCGT-3'. All Northern blots were
reprobed with random primed 32P-labeled human G3PDH
cDNA to confirm equivalent loading of RNA samples.
Western Analysis--
Samples for Western analysis were prepared
using 400 µl of radioimmune precipitation extraction buffer/100-mm
plate. The protein concentration for each sample was determined by
Bradford analysis. Equal amounts of total protein were separated on a
14% SDS-polyacrylamide minigel, and proteins were transferred to
polyvinylidene difluoride membranes. Membranes were treated for a
minimum of 2 h with specific primary antibody followed by
incubation with secondary antibody conjugated to horseradish
peroxidase. Visualization was carried out with the ECL Plus detection kit.
Hypodiploidy Apoptosis Assay--
Cells were treated with
oligonucleotides and apoptosis inducers as described. Both floating and
adherent cells were harvested 24-48 h after treatment; washed once
with 1 ml of PBS, 5 mM EDTA; and fixed with 1 ml of 70%
EtOH while vortexing gently. Fixed cells were stored at 4 °C for
1 h to several days. Cells were pelleted by centrifugation, washed
once with 1.0 ml of PBS, 5 mM EDTA, and resuspended with
0.3-1.0 ml Propidium Iodide mix (250 µg/ml propidium iodide, 5 µg/ml RNase A, 1 × PBS, and 5 mM EDTA). After
incubation in the dark for 1 h at room temperature, the cells were
analyzed on a Becton Dickinson FACscan, and apoptotic cells
(sub-G1 population) were quantified.
Mitochondrial Transmembrane Potential (
Ym)--
To evaluate
the
Ym, cells were incubated with the cationic lipophilic dye
MitoTracker orange CMTMRos (150 nM) for 15 min at 37 °C
in the dark (20). Control cells were simultaneously treated with 50 µM of the protonophore, carbonyl cyanide
m-chlorophenylhydrazone, which disrupts
Ym. Both adherent
and floating cells were collected, washed once with PBS, 2% bovine
serum albumin, and fixed in 1 ml of PBS containing 4% paraformaldehyde
for 15 min while shaking at room temperature. Fixed cells were stored
in the dark at 4 °C for 1 day prior to analysis by flow cytometry.
The percentage of cells with decreased fluorescence, and therefore with
reduced
Ym (compared with control cells not treated with
oligonucleotides or apoptotic inducers), were determined and indicated
on the bar graphs as percentage of cells gated. Cells treated with
carbonyl cyanide m-chlorophenylhydrazone generally gave
greater than 70% cells gated and showed a shift of 0.5 log units to a
lower fluorescence.
Caspase 3 Activity--
Caspase 3 activity was measured using
Caspase 3 colorimetric assay kit from CLONTECH
(Palo Alto, CA). Briefly, whole cell lysates were obtained, protein
concentrations were determined, and equal amounts of protein were added
to 50 µM substrate
(Asp-Glu-Val-Asp-p-nitroanilide) in assay buffer. Samples
were incubated at room temperature and analyzed using a
spectrophotometer (OD 418).
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RESULTS |
Bcl-2 Family Members in HUVEC--
Ribonuclease protection assay
was utilized to identify Bcl-2 family members present in basal and
TNF-
-treated HUVEC. High levels of the antiapoptotic members Bcl-XL
and Mcl-1 were observed in both basal and induced cells, while Bcl-2
was not detected (Fig. 1). In contrast,
the antiapoptotic member A1 was found in low abundance in basal HUVEC
but increased ~10-fold following TNF-
treatment. Maximal induction
of A1 was observed ~4 h after treatment. Interestingly, Bcl-XL was
down-regulated approximately 20% upon treatment with TNF-
.

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Fig. 1.
Bcl-2 family members present in basal and
TNF- -induced HUVEC. Total RNA was
extracted from basal HUVEC or cells treated with TNF- (10 ng/ml) for
4 h. The ribonuclease protection assay was performed according to
the supplier's instructions. hGAPDH, human
glyceraldehyde-3-phosphate dehydrogenase.
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Inhibition of A1 mRNA through Antisense Oligonucleotide
Treatment--
An effective antisense oligonucleotide directed toward
A1 was identified from a screen targeting 14 distinct positions on the
A1 mRNA, extending from the 5'-UTR to the 3'-UTR using methods as
described previously (21). All oligonucleotides were 20 nucleotides in
length containing chimeric 2'-O-methoxyethyl modifications on a mixed backbone of phosphodiester and phosphorothioate linkages (Table I). The most effective A1
antisense oligonucleotide was directed toward the 3'-UTR, immediately
downstream from the stop codon, and was utilized in all subsequent
experiments.
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Table I
Sequence and chemistry of oligonucleotides
All oligonucleotides utilized were chimeras with 2'-deoxy or
2'-O-methoxyethyl modifications. Sequences containing the
2'-O-methoxyethyl modifications are shown in parentheses.
The A1 antisense oligonucleotide and its control contained
phosphodiester linkages in the 2'-O-methoxyethyl regions and
phosphorothioate linkages in the 2'-deoxynucleotide region (as
indicated by O or S). The Bcl-XL antisense oligonucleotide and its
control contained phosphorothioate linkages
throughout.
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Fig. 2 shows the
concentration-dependent decrease of the A1 mRNA with
increasing amounts of A1 antisense oligonucleotide, while a mismatch
control oligonucleotide failed to show a reduction, demonstrating a
sequence specific inhibition. The apparent IC50 for the A1
antisense compound was less than 10 nM. Treatment of HUVEC
with 50 nM of the A1 antisense oligonucleotide reduced the A1 mRNA to basal levels, completely inhibiting the TNF-
-induced component. Reductions in A1 mRNA to basal levels were observed up
to 30 h after antisense treatment (data not shown).

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Fig. 2.
Dose-dependent inhibition of A1
mRNA by antisense oligonucleotide. HUVEC were incubated with
increasing concentrations of a specific A1 antisense or mismatch
oligonucleotide. After a recovery period of 16 h, cells were
induced with TNF- for 4 h, and total RNA was extracted.
Northern analysis was carried out, and the membrane was probed
sequentially with a 32P-labeled A1 probe and a G3PDH
32P-labeled probe (shown in the bottom
panel). The amount of specific RNA in each lane was
quantified. The graph shows the average of three separate
experiments.
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Inhibition of Bcl-XL mRNA and Protein through Antisense
Oligonucleotide Treatment--
A potent, sequence-specific antisense
oligonucleotide directed toward Bcl-XL has previously been identified
and shown to decrease Bcl-XL mRNA and protein levels in both human
keratinocytes and A549
cells.2 Here we tested its
ability to inhibit Bcl-XL mRNA in HUVEC. As shown in Fig.
3, treatment of HUVEC with 100 nM of the Bcl-XL oligonucleotide decreased the Bcl-XL
mRNA levels to ~5% of control, with an apparent IC50
less than 20 nM. In comparison, a mismatch control
oligonucleotide did not reduce the Bcl-XL mRNA levels. The Bcl-XL
antisense compound is targeted to the coding region of the gene and is
a chimera of 2'-O-methoxyethyl modification with a
phosphorothioate backbone (Table I). It should be noted that the Bcl-XL
antisense oligonucleotide utilized here targets both the long and short
forms of Bcl-X (Bcl-XL and Bcl-XS). However, HUVEC contain such low
levels of Bcl-XS mRNA, either basally or after induction with
TNF-
(see Fig. 1), that the contribution of Bcl-XS to the total
Bcl-X mRNA levels detected in our experiments is insignificant.

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Fig. 3.
Dose-dependent inhibition of
Bcl-XL mRNA by antisense oligonucleotide. HUVEC were incubated
with the increasing concentrations of a specific Bcl-XL antisense or
mismatch oligonucleotide. Cells were harvested 24 h later, and
total RNA was extracted. Northern analysis was carried out, and the
membrane was probed sequentially with a Bcl-XL 32P-labeled
probe and a G3PDH 32P-labeled probe (shown in the
bottom panel). The amount of specific RNA in each
lane was quantified, and the figure shows results from a
typical experiment.
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Bcl-XL protein expression was also examined by Western analysis. As
shown in Fig. 4, cells treated with the
Bcl-XL antisense compound showed a time-dependent decrease
in Bcl-XL protein levels that was not observed in cells treated with
the control compound. Maximal reduction was observed 48 h after
oligonucleotide treatment. In our experiments, the Bcl-XL protein
migrated as a doublet (visible only after significant reductions in
protein), and both bands decreased with antisense oligonucleotide
treatment. The observed doublet could represent changes in the
phosphorylation state of Bcl-XL. If the Bcl-XS protein were
present, it should migrate well below the Bcl-XL band under our
conditions. Thus, the Bcl-XL antisense compound was able to decrease
Bcl-XL protein levels to roughly 5% of amounts in untreated cells.

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Fig. 4.
Inhibition of Bcl-XL protein expression.
Cells were treated with Bcl-XL antisense or mismatch oligonucleotide
(50 nM) and harvested at 16, 24, or 48 h. Bcl-XL
protein levels were determined by Western blot. The Bcl-XL band
migrates at ~30 kDa.
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Specificity of Antisense Oligonucleotide Treatment--
In order
to demonstrate the specificity of A1 and Bcl-XL antisense
oligonucleotides, HUVEC were treated with A1 antisense oligonucleotide,
A1 control, Bcl-XL antisense oligonucleotide, or Bcl-XL control, in the
presence or absence of TNF-
. Northern analysis was carried out, and
the membrane was probed sequentially with A1, Bcl-XL, and
G3PDH-specific probes. The A1 antisense compound was effective in
reducing A1 mRNA without affecting the Bcl-XL mRNA. Likewise,
the Bcl-XL antisense compound specifically reduced Bcl-XL mRNA
without affecting A1 mRNA (Fig. 5).
In addition, there was no affect on either target with the control
oligonucleotides. These results demonstrate that the antisense
oligonucleotides utilized here can specifically inhibit the target
mRNA without affecting other Bcl-2 family members.

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Fig. 5.
Specificity of antisense oligonucleotide
treatment. Cells were treated with 50 nM of A1
antisense, A1 control, Bcl-XL antisense, or Bcl-XL control
oligonucleotides, followed by a recovery period of 24 h. Indicated
cells were treated with TNF- for 4 h, and all cells were
harvested for total RNA. Northern analysis was carried out, and the
membrane was probed sequentially with A1-, Bcl-XL-, and G3PDH-specific
probes.
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HUVEC Viability after Inhibition of Endogenous A1 and Bcl-XL
Protein: Basal Versus TNF-
Induction--
Because A1 is induced by
TNF-
treatment, its role in protecting HUVEC from TNF-
-induced
apoptosis has been suggested (12). To address this point, cells were
treated with 50 nM A1 antisense oligonucleotide or control
oligonucleotide and subsequently induced with TNF-
. Apoptotic cells
with fragmented DNA were identified by flow cytometry analysis. As
shown in Fig. 6A, inhibition
of the TNF-
-induced A1 mRNA did not significantly affect the
number of apoptotic cells observed after TNF-
treatment. However,
cells treated with a combination of TNF-
and cycloheximide, which
prevents the up-regulation of all TNF-
-inducible cytoprotective
genes, resulted in over 50% cell death under these conditions. These results suggest that A1 is not solely responsible for protection of
HUVEC from TNF-
-induced programmed cell death.

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Fig. 6.
Effect of A1 and Bcl-XL inhibition on DNA
fragmentation (basal versus TNF-
induction). A, HUVEC were treated with Lipofectin
alone ( ), 50 nM A1 antisense ( ), or 50 nM
control oligonucleotide ( ) as indicated. Cells were allowed to
recover for 16 h, induced with TNF- , cycloheximide (10 µg/ml), or TNF- plus cycloheximide, followed by an additional 24-h
incubation. Cells were harvested and analyzed by fluorescence-activated
cell sorting for hypodiploidy as described under "Materials and
Methods." B, HUVEC were treated with Lipofectin alone
( ), 50 nM Bcl-XL antisense oligonucleotides ( ), or
control oligonucleotide ( ) as indicated. After a 24-h period,
TNF- was added to the indicated cells, followed by an additional
24-h incubation. C, HUVEC were treated with Lipofectin alone
( ), combinations of Bcl-XL- and A1-specific antisense
oligonucleotides (50 nM/each) ( ), or control
oligonucleotides (50 nM/each) ( ). Cells were allowed to
recover for 24 h, induced with TNF- as indicated and incubated
for an additional 24-h period. Results shown are average of duplicate
samples.
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Similar experiments were also carried out with the Bcl-XL antisense
compound. Inhibition of Bcl-XL protein in basal HUVEC caused 10-25%
of the cell population to undergo apoptosis (Fig. 6B). Thus,
Bcl-XL may play an important role in the basal viability of endothelial
cells. On the other hand, inhibition of the Bcl-XL protein did not
render cells more sensitive to TNF-
treatment (i.e. the
difference between Bcl-XL and control remained the same in the presence
of TNF-
), indicating that Bcl-XL is not critical in protecting cells
from this apoptotic stimuli.
Because inhibition of either A1 or Bcl-XL protein did not render HUVEC
more susceptible to TNF-
induced apoptosis, we investigated whether
simultaneous inhibition of both proteins would result in increased
apoptosis. Therefore, HUVEC were treated with both A1 and Bcl-XL
antisense compounds, allowed to recover for 24 h, and then treated
with or without TNF-
. Fig. 6C shows that simultaneous inhibition of A1 and Bcl-XL did not significantly render HUVEC more
susceptible to TNF-
treatment.
Ability of A1 Antisense Oligonucleotide and Bcl-XL Antisense
Oligonucleotide to Sensitize HUVEC to Treatment with Ceramide or
Staurosporin: Effect on DNA Fragmentation and Mitochondrial
Transmembrane Potential--
Next we tested the sensitivity of HUVEC
treated with the A1 or Bcl-XL antisense oligonucleotide to treatment
with the protein kinase inhibitor staurosporin or the lipid second
messenger ceramide. Both of these agents have been shown to induce
apoptosis in many cell types including endothelial cells (22, 23).
Treatment of HUVEC with 1 nM staurosporin or 5 µM C6-ceramide alone did not significantly affect the
cellular DNA fragmentation. However, cells with decreased levels of
Bcl-XL protein showed increased sensitivity toward these low
concentrations of apoptotic stimuli (Figs.
7 and
8A). In contrast, cells
pretreated with the A1 antisense compound showed only a slight increase
in DNA fragmentation after treatment with TNF-
in the presence of
either staurosporin or ceramide (Fig.
9A). As shown in Fig.
9A, HUVEC were more sensitive to ceramide treatment when
carried out in the presence of TNF-
as described previously (23).

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Fig. 7.
Bcl-XL inhibition sensitizes cells to
treatment with staurosporin or ceramide. Cells were treated with
Lipofectin reagent alone, 50 nM Bcl-XL antisense, or 50 nM control. After 24 h, cells were treated with either
staurosporin (1 nM) or ceramide (5 µM)
followed by an additional 24-h incubation. Harvested cells were
analyzed by fluorescence-activated cell sorting for hypodiploidy.
Percentages of apoptotic cells are indicated.
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Fig. 8.
Effect of Bcl-XL inhibition, ceramide,
and staurosporin on DNA fragmentation and Ym
in the presence and absence of Z-VAD.fmk. Cells were treated with
Lipofectin reagent alone ( ), 50 nM Bcl-XL antisense
( ), or 50 nM control ( ) as indicated. After 24 h, cells were treated with ceramide (10 µM), staurosporin
(2 nM), or Z-VAD.fmk (100 µM) as indicated,
followed by an additional 24-h incubation. Harvested cells were
analyzed by fluorescence-activated cell sorting for hypodiploidy
(A and B) or Ym (C and
D) as described under "Materials and Methods." Results
shown are representative of five independent experiments.
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Fig. 9.
Effect of A1 inhibition, ceramide, and
staurosporin on DNA fragmentation and Ym in
the presence and absence of Z-VAD.fmk. Cells were treated with
Lipofectin reagent alone ( ), 50 nM A1 antisense ( ),
or 50 nM control ( ) as indicated. After 16 h, cells
were induced with TNF- and subsequently treated with ceramide (5 µM), staurosporin (1 nM), or Z-VAD.fmk (100 µM) followed by an additional 24-h. incubation. Harvested
cells were analyzed by fluorescence-activated cell sorting for
hypodiploidy (A and B) or Ym (C and
D) as described under "Materials and Methods." Results
shown are representative of five independent experiments.
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One of the major mechanisms of Bcl-2-mediated cytoprotection is by
protecting mitochondrial function (reviewed in Refs. 24 and 25).
Therefore, we investigated the effect of A1 and Bcl-XL inhibition on
the
Ym. A reduction in
Ym has been shown to accompany apoptosis
in many experimental models and can be monitored by the
potential-sensitive, fluorescent dye CMTMRos. To confirm that CMTMRos
was sensitive to mitochondrial transmembrane depolarization, control
cells were treated with 50 µM of the mitochondrial
uncoupling agent carbonyl cyanide m-chlorophenylhydrazone.
As shown in Fig. 8C, treatment of HUVEC with Bcl-XL
antisense compound resulted in a reduction in the
Ym. This reduction
was potentiated by the combination of Bcl-XL inhibition and treatment
with either staurosporin or ceramide. Thus, endogenous Bcl-XL may
function by maintaining the mitochondrial integrity of resting HUVEC,
where loss of Bcl-XL results in greater susceptibility to mitochondrial
dysfunction. In contrast to these results, inhibition of A1 did not
effect the
Ym, after treatment with either TNF-
alone or TNF-
in combination with ceramide or staurosporin (Fig. 9C).
Involvement of Caspases in HUVEC Apoptosis Resulting from Bcl-XL
Inhibition--
HUVEC were treated with the A1 or Bcl-XL antisense
oligonucleotides or controls, in the presence or absence of Z-VAD.fmk, a broad-based cell-permeable caspase inhibitor. As shown in Fig. 8B, Z-VAD.fmk completely inhibited the DNA fragmentation
resulting from Bcl-XL inhibition alone or from Bcl-XL inhibition in the presence of ceramide or staurosporin. Similar results were also obtained using Z-DEVD.fmk, a known inhibitor of caspases 3, 7, and 8 (data not shown). Besides affecting DNA fragmentation, Z-VAD.fmk also
inhibited
Ym loss induced by Bcl-XL antisense oligonucleotide or
Bcl-XL antisense oligonucleotide and staurosporin (Fig. 8D). However, inhibition of
Ym was not observed when cells deficient in
Bcl-XL protein were treated with ceramide. Bcl-XL antisense oligonucleotide plus ceramide-treated cells were not viable as shown by
PI exclusion (data not shown), although DNA fragmentation was
completely prevented.
Consistent with caspase inhibition data, proteolysis of procaspase 3 was also observed in Bcl-XL-deficient cells and cells treated with
staurosporin. This reduction in procaspase 3 was inhibited by Z-VAD.fmk
(Fig. 10A). In addition,
separate experiments demonstrated an increase in caspase 3 activity
following inhibition of Bcl-XL or Bcl-XL inhibition in the presence of
staurosporin. The increased caspase 3 activity correlated with the
proteolysis of procaspase 3 observed in the Western blot (Fig.
10B).

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Fig. 10.
Inhibition of Bcl-XL results in procaspase 3 cleavage and increased caspase 3 activity that is inhibited by
Z-VAD.fmk. Cells were treated with Bcl-XL-specific or control
antisense oligonucleotide (50 nM), and 24 h later were
treated with staurosporin and/or Z-VAD.fmk. After an additional 24-h
incubation, whole cell lysates were harvested, and protein
concentrations were determined. A, procaspase 3 protein
levels were determined by Western blot, loading equal amounts of
protein in each lane. B, in a separate experiment, caspase 3 activity was determined using 50 µM
DEVD-p-nitroanilide substrate and recording OD 418.
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DISCUSSION |
To date, much work has been carried out studying the protective
role of Bcl-2 and Bcl-XL, while relatively little is known about the
other antiapoptotic Bcl-2 family members A1, Bcl-W, and Mcl-1. One
unresolved question has been the specific function or molecular
mechanism of each isoform, since many cells express more than one.
Through ribonuclease protection assay analysis, we show that
endothelial cells express Bcl-XL and Mcl-1 constitutively, while A1 is
found in very low amounts in basal cells but can be up-regulated by
TNF-
. Therefore, we initiated studies aimed at addressing and
comparing the normal physiological role between two of these family
members, A1 and Bcl-XL, by utilizing specific antisense
oligonucleotides to inhibit the endogenous levels of each protein.
Although the identity of the survival factor(s) responsible for halting
the TNF-
death pathway remains to be firmly established, a number of
recent reports have identified several candidates. One such candidate,
A1, was originally identified in phorbol ester-stimulated endothelial
cells and shown to be induced by the inflammatory cytokines TNF-
and
interleukin-1 (27). Further experiments demonstrated that endothelial
cells expressing high levels of A1, through retrovirus-mediated
transfer, were protected against cell death initiated by TNF-
plus
actinomycin D (12). This protection was also observed with
overexpression of Bcl-XL (12). However, others have reported that
overexpression of either A1 or Bcl-XL did not rescue HUVEC from death
induced by TNF-
plus cycloheximide, although both effectively
inhibited apoptosis induced by TNF-
plus ceramide. (23). To help
clarify the role of A1 as a TNF-
-inducible cytoprotective gene, we
asked whether inhibition of A1 would render cells sensitive to
TNF-
-induced apoptosis. We demonstrate that treatment of HUVEC with
a highly specific and potent antisense oligonucleotide directed toward
A1 completely inhibited the TNF-
-inducible component of the A1
mRNA. However, this inhibition did not render cells significantly
more susceptible to apoptosis by TNF-
. Furthermore, cells with
decreased levels of Bcl-XL, or both Bcl-XL and A1, continued to show
resistance to TNF-
treatment. Thus, A1 and Bcl-XL do not appear to
play critical roles in protecting endothelial cells from TNF-
.
In contrast, inhibition of the endogenous levels of Bcl-XL resulted in
a substantial amount of cell death in basal cells. In addition,
Bcl-XL-deficient cells were rendered sensitive to concentrations of
ceramide and staurosporin that were without effect in cells expressing
normal levels of Bcl-XL. Thus, Bcl-XL may function physiologically as a
protective gene that, under normal resting conditions, helps
endothelial cells maintain their quiescent phenotype and perform their
normal barrier functions even in the presence of low levels of
stimulants (26). This would be critical for endothelial cells in
vivo, which are constantly assaulted by noxious compounds such as
oxidized low density lipoproteins or cytokines. Our results are
consistent with overexpression data indicating that Bcl-XL protects
cells from assault from a wide range of apoptotic stimulants, including
ceramide (12, 28) and staurosporin (29). It is interesting to note that
as the concentrations of staurosporin and ceramide increased, the
differences observed between normal and Bcl-XL-inhibited HUVEC slowly
disappeared (data not shown). These results suggest that endogenous
Bcl-XL can protect HUVEC from apoptotic stimuli up to a threshold level after which its presence or absence does not effect the outcome. This
may make sense physiologically, since a high level of assault, causing
substantial damage to a cell, would be desirable to end in cell death.
One of the major mechanisms of Bcl-2-mediated cytoprotection is by
protecting mitochondrial function (reviewed in Refs. 24 and 25). More
specifically, these proteins are thought to function through
maintaining the mitochondrial permeability transition pore opening (and
therefore protecting
Ym) and preventing release of the caspase
activators, AIF and cytochrome c, into the cytosol (25). In
support of these functions, several groups have now reported that
overexpression of Bcl-XL or Bcl-2 confers protection upon mitochondria,
making it more difficult for many stimuli to induce PT pore opening and
release of AIF and cytochrome c (25, 30-32).
Consistent with a role in the maintenance of mitochondria integrity, we
show that inhibition of endogenous Bcl-XL protein levels caused a
reduction in
Ym. This reduction was further potentiated with the
combination of Bcl-XL inhibition plus ceramide or staurosporin. However, the observed
Ym loss showed different responses to caspase inhibitors. In cells treated with Bcl-XL antisense oligonucleotide or
Bcl-XL antisense oligonucleotide plus staurosporin, Z-VAD.fmk prevented
apoptosis, within the time frame studied, as shown by a lack of DNA
fragmentation and maintenance of
Ym. However, Z-VAD.fmk was unable
to protect cells against
Ym loss and subsequent death induced by
Bcl-XL antisense oligonucleotides plus ceramide, although DNA
fragmentation was completely blocked.
These results can be explained in view of the current models. When
cytochrome c is released into the cytosol, it can bind to
Apaf-1, resulting in the activation of caspase 9 and subsequently caspase 3. It is now apparent that in some systems, cytochrome c release occurs prior to loss in
Ym and that the
subsequent activation of caspases is required for
Ym loss, resulting
in a circular amplification loop (Refs. 30 and 33-35; reviewed in Ref.
36). Thus, in this model, disruption of
Ym is prevented by caspase
inhibitors as was observed with Bcl-XL inhibition and Bcl-XL inhibition
plus staurosporin.
Alternatively, in a number of other models, inhibition of caspases with
Z-VAD.fmk fails to prevent the disruption of mitochondrial membrane
function, despite the fact that Z-VAD.fmk does prevent the acquisition
of the many biochemical hallmarks including DNA fragmentation (37-40).
Thus, in these systems caspase activation is downstream of mitochondria
dysfunction, and furthermore, mitochondrial disruption is sufficient to
cause cell death, through apoptosis or necrosis. This model would be
consistent with our observations with Bcl-XL inhibition plus ceramide.
Furthermore, a recent report demonstrated that GD3 ganglioside
synthesis, which can be triggered by cell-permeating ceramides,
resulted in disruption of
Ym and induced apoptosis in a
caspase-independent fashion (37).
Taken together, our data are consistent with endogenous Bcl-XL
functioning by maintaining mitochondrial integrity of resting HUVEC,
where loss of Bcl-XL results in greater susceptibility to mitochondrial
dysfunction. The protection of
Ym afforded by Bcl-XL functions in
both caspase-dependent and caspase-independent mechanisms.
These results demonstrate a definitive role for Bcl-XL in these
processes and are complementary to overexpression data obtained previously.