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Blood, Vol. 93 No. 7 (April 1), 1999:
pp. 2353-2359
Subcellular Distribution and Redistribution of Bcl-2 Family Proteins in
Human Leukemia Cells Undergoing Apoptosis
By
Li Jia,
Marion G. Macey,
Yuzhi Yin,
Adrian C. Newland, and
Stephen
M. Kelsey
From the Department of Haematology, St Bartholomew's and the Royal
London School of Medicine and Dentistry, London, UK.
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ABSTRACT |
It has been suggested that the ratio of Bcl-2 family proapoptotic
proteins to antiapoptotic proteins determines the sensitivity of
leukemic cells to apoptosis. However, it is believed that Bcl-2 family
proteins exert their function on apoptosis only when they target to the
mitochondrial outer membrane. The vinblastine-resistant T-lymphoblastic
leukemic cell line CEM/VLB100 has increased sensitivity to
tumor necrosis factor- (TNF-)-induced cytochrome c
release, mitochondrial respiratory inhibition, and consequently
apoptosis, compared with parental CEM cells. However, there was no
difference between the two cell lines in the expression of Bcl-2 family
proteins Bcl-2, Bcl-XL, Bcl-XS, Bad, and Bax at
the whole cell level, as analyzed by Western blotting. Bcl-2 mainly
located to mitochondria and light membrane as a membrane-bound protein,
whereas Bcl-XL was located in both mitochondria and
cytosol. Similar levels of both Bcl-2 and Bcl-XL were
present in the resting mitochondria of the two cell lines. Although the
proapoptotic proteins Bcl-XS, Bad, and Bax were mainly
located in the cytosol, CEM/VLB100 mitochondria expressed
higher levels of these proapoptotic proteins. Subcellular redistribution of the Bcl-2 family proteins was detected in a cell-free
system by both Western blotting and flow cytometry after exposure to
TNF-. The levels of Bcl-2 family proteins were not altered at the
whole cell level by TNF-. However, after exposure to TNF-, Bax,
Bad, and Bcl-XS translocated from the cytosol to the
mitochondria of both cell lines. An increase in Bcl-2 levels was
observed in CEM mitochondria, which showed resistance to
TNF--induced cytochrome c release. By contrast, decreased
mitochondrial Bcl-2 was observed in CEM/VLB100 cells, which
released cytochrome c from the mitochondria and underwent
apoptosis as detected by fluorescence microscopy. We conclude that
mitochondrial levels of Bcl-2 family proteins may determine the
sensitivity of leukemic cells to apoptosis and that, furthermore, these
levels may change rapidly after exposure of cells to toxic stimuli.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
Bcl-2, Bcl-X (Bcl-XL and
Bcl-XS), Bad, and Bax are members of the Bcl-2 family
proteins that play important roles in regulating cell survival and
apoptosis. Antiapoptotic members such as Bcl-2 and Bcl-XL
prevent apoptosis in response to a wide variety of
stimuli.1-7 Conversely, proapoptotic proteins, Bad, Bax,
and Bcl-XS, can accelerate death and in some instances are sufficient to cause apoptosis.8-12 The ratio of Bcl-2
family proapoptotic to antiapoptotic proteins dictates the
susceptibility of cells to a variety of apoptotic
stimuli13,14 and is an important determinant of the
sensitivity of leukemic cells to apoptosis induced by chemotherapeutic
or immunotherapeutic agents.15-17
A major pathway of apoptosis has been shown that is controlled at the
mitochondrial level. During apoptosis, cytochrome c, which is
normally located in the mitochondrial intermembrane space, is released.
Together with other cytosolic factors, cytochrome c can trigger
the activation of caspase-3 (CPP32) in a cell-free system.1,2,18-20 Functional Bcl-2 family proteins exert
many of their effects when they locate to the mitochondrial outer
membrane. Overexpression of Bcl-2 or Bcl-XL inhibits
apoptosis by blocking the release of cytochrome
c.1-4 Bax targeted to mitochondria can trigger
rapid release of cytochrome c.7,21 However, the expression of Bcl-2 family proteins at the mitochondrial level has not
been considered as an important determinant of the sensitivity of
leukemic cells to apoptosis.
Bcl-2, Bcl-XL, Bcl-XS, and Bax possess a
carboxyterminal transmembrane (TM) region that is believed to serve as
a membrane anchor. However, Bad does not contain a TM
region.22 As a result Bcl-2, Bcl-XL, and
Bcl-XS locate to the mitochondrial outer membrane, nuclear
envelope, and endoplasmic reticulum.10,23-25 However, Bcl-XL and Bcl-XS have also been identified in
the cytosol.10,11 It has been suggested that Bax targets to
organelle membranes,26 and in particular to
mitochondria,27 by its TM region. It has been recently
observed that Bax is present predominantly in the cytosol in the
resting state and moves to mitochondria during exposure to proapoptotic
stimuli.11,12 Although Bad does not contain a TM region,
unphosphorylated Bad can heterodimerize with Bcl-XL at
mitochondrial membrane sites to promote cell death.28,29
We have previously shown that the vinblastine-resistant cell
line, CEM/VLB100, has increased sensitivity to
TNF- -induced mitochondrial respiratory inhibition, mitochondrial
ultracondensation, and consequently apoptosis, compared
with parental CEM cells.30-32 In this study, we examined
the ratio of Bcl-2 family proapoptotic to antiapoptotic proteins at
both whole cell and mitochondrial levels. We postulate that the
sensitivity of leukemic cells to apoptosis can be determined by Bcl-2
family protein expression at the mitochondrial level. We describe that
cells reset the ratio of mitochondrial proapoptotic protein to
antiapoptotic protein in response to TNF-.
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MATERIALS AND METHODS |
Materials.
Monoclonal mouse anti-Bcl-2 (100), polyclonal rabbit
anti-Bcl-XS/L (S-18), and polyclonal rabbit anti-Bax
(P-19) antibodies were from Santa Cruz Biotechnology Inc (Santa Cruz,
CA). Monoclonal mouse anti-Bad antibody (48) was from Transduction Lab
(Lexington, KY). Native monoclonal mouse anti-cytochrome c
antibody (6H2.B4) was purchased from PharMingen (San Diego, CA).
MitoTracker red CMXRos and 3,3'-dihexyloxacarbocyanine iodide
(DiOC6[3]) were from Molecular Probes Inc (Eugene, OR).
Monoclonal mouse anti- -actin (AC-74) antibody, TNF-, DAPI,
carbonyl cyanide m-chlorophenylhydrazone (CCCP), and all the chemicals
were from Sigma (Poole, UK).
Cell lines and the preparation of subcellular fractions.
The human T-lymphoblastic CEM and its vinblastine-resistant subclone
CEM/VLB100 leukemia cell lines were used in this study. Cell culture was as previously described.30 Leukemia cells
(5 × 107) were washed with Ca2+ and
Mg2+-free phosphate-buffered saline (PBS) and resuspended
in 1 mL of buffer A (250 mmol/L sucrose, 10 mmol/L HEPES-KOH, pH 7.4, 2 mmol/L NaCl, 2.5 mmol/L KH2PO4, 0.5 mmol/L
EGTA, 2 mmol/L MgCl2, 5 mmol/L pyruvate, 1 mmol/L DTT, 0.1 mmol/L phenylmethylsulfonyl fluoride [PMSF], 20 µmol/L cytochalasin
B, 2 mmol/L adenosine triphosphate [ATP], 10 mmol/L phosphocreatine,
50 µg/mL creatine phosphokinase) and incubated for 20 minutes on ice.
Cells were then broken with a glass Dounce homogenizer (Jencons,
Leighton Buzzard, UK). After pelleting the nuclei, 790g for 10 minutes at 4°C, the post-nuclear supernatant was further spun at
10,000g for 10 minutes at 4°C. The crude mitochondrial
pellet was purified by passing a gradient sucrose (0.1 to 0.3 mol/L)
cushion at 9,000g for 8 minutes to purify mitochondria. The
purity of mitochondria was determined by Western blotting as being
-actin negative. The supernatant (S10 fraction) was further
ultracentrifuged at 16,000g and then filtered by passing
through a 0.22-µm Ultrafilter (Sigma) to get purified cytosol. Light
membrane was obtained from a pellet of S10 fraction and the membrane on
the ultrafilter, which was identified as the RNA positive and
cytochrome c negative fraction. Protein concentration was
measured using Bradford reagent (Bio-Rad, Hertfordshire, UK).
Western blotting.
Whole cells, purified mitochondria, or light membrane was lysed in
lysis buffer (1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium
dodecyl sulfate (SDS), 1 mmol/L PMSF, 1 mmol/L DTT, 10 µg/mL
aprotinin, 10 µg/mL leupeptin, 1 mmol/L sodium orthovanadate in PBS,
pH 7.4) for 30 minutes at 4°C. Lysed cells or mitochondria were
then centrifuged at 16,000g for 30 minutes at 4°C. Protein extracts were further mixed with sample buffer (187.5 mmol/L Tris-HCl, pH 6.8, 6% SDS, 15% -mercaptoethanol, 30% glycerol, 0.006%
bromophenol blue) and heated at 100°C for 3 minutes. Fifty
micrograms of protein was subjected to 12% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, followed by
transfer to nitrocellulose membrane (Bio-Rad). The blot was blocked by
5% nonfat milk and probed with anti-Bcl-2 antibody (1:100 dilution),
anti-Bcl-XS/L antibody (1:200 dilution),
anti-Bad antibody (1:500 dilution), or anti-Bax (1:200 dilution) and
anti--actin (1:10,000 dilution) antibodies. Secondary probes
constituted horseradish peroxidase (HRP)-labeled anti-mouse antibody
(1:2,000 dilution, Santa Cruz) or anti-rabbit antibody (1:3,000
dilution, Santa Cruz). Filters were washed three times with 0.05%
Tween-20 (for monoclonal anti-mouse antibody) or 0.1% Tween-20 (for
polyclonal anti-rabbit antibody) containing PBS. Specific protein
complexes were identified using the "SuperSignal" enhanced
chemiluminescence (ECL) reagent (Pierce, Rockford, IL). Rainbow markers
(Amersham, Little Chalfont, UK) served as standard molecular weights.
Relative densitometry analysis of autoradiographs was based on
integrated density percentage value using an AlphaImager 2000 scanner
fitted with AlphaEase Stand Alone software (Alpha Innotech Corp, San
Jose, CA).
Flow cytometry of isolated mitochondria.
To measure Bcl-2 expression, mitochondria were blocked by incubation
with 1% bovine serum albumin (BSA) in buffer B (225 mmol/L mannitol,
75 mmol/L sucrose, 10 mmol/L KCl, 10 mmol/L Tris-HCl, 5 mmol/L
KH2PO4, pH 7.2) for 30 minutes on ice and
washed with buffer B. Twenty microliters of mitochondrial suspension in
buffer B (containing 0.1% BSA) was then incubated with 0.5 µg of
primary anti-Bcl-2 antibody or 0.5 µg of control antibody for 30 minutes on ice. After one wash, mitochondria were resuspended with 0.5 µg of fluorescein isothiocyanate (FITC)-conjugated anti-mouse antibody and incubated for 30 minutes on ice, washed, and resuspended in 1mL of buffer B for flow cytometry analysis using the Lysys II
software package on a FACScan flow cytometer (Becton Dickinson, Oxford,
UK). To measure mitochondrial membrane potential (  m), mitochondria were suspended in buffer B and incubated with 80 nmol/L
DiOC6(3) for 15 minutes at 37°C followed by analysis on a FACScan flow cytometer.
Immunofluorescence analysis of cytochrome c release.
To colocalize cytochrome c in mitochondria, intact cells were
first labeled with the mitochondrion-specific dye, MitoTracker red
CMXRos. Cells in culture medium were incubated with MitoTracker CMXRos
(100 nmol/L for the CEM cell line, 150 nmol/L for the
CEM/VLB100 cell line) at 37°C for 30 minutes. Cells
were washed twice with Ca2+/Mg2+-free PBS and
resuspended in 10% FCS containing culture medium. One hundred fifty
microliters of cell suspension (5 × 105 cells/mL) was
cytocentrifugated onto a microscope slide using a Shandon Southern
Cytospin (Pittsburgh, PA) centrifuge at 1,200 rpm for 3 minutes. Slides
were air dried, permeabilized, and fixed in 4% paraformaldehyde/0.05%
saponin in Ca2+/Mg2+-free PBS for 15 minutes.
Cells were then washed twice in 0.03% saponin containing PBS and
incubated in a blocking solution (1% BSA, 1% normal goat serum, and
0.1% Tween-20 in PBS) for 30 minutes. After washing once, cells were
incubated with the anti-cytochrome c antibody 6H2.B4 (25 µg/mL) diluted in blocking solution for 2 hours at room temperature
in a humidified chamber. Cells were washed in PBS then incubated with
FITC-conjugated anti-mouse secondary antibody (Sigma) at a 1:10
dilution in blocking solution for 1 hour in the dark. Cells were rinsed
three times in PBS, then incubated with 50 ng/mL DAPI-containing PBS
for 1 minute. Slides were air dried at 4°C in the dark and mounted
in Immuno-Mount solution (Shandon) and viewed under a Zeiss Axioskop
fluorescence microscope (Zeiss, Germany) attached to a CCD camera
(Photometric Ltd, Tucson, AZ) driven by IPLLabs Spectrum
and SmartCapture (Cambridge, UK) software. The filter wheel was set at
Texas red (excitation 540 to 580 nm/emission 600 to 660 nm),
fluorescein (excitation 465 to 495 nm/emission 515 to 555 nm), and DAPI
(excitation 310 to 380 nm/emission 435 to 485 nm). Photographs for the
triple labeling experiments were taken through triple exposure.
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RESULTS |
The expression of Bcl-2 family proteins in TNF-sensitive and
TNF-resistant cell lines at the whole cell level.
We initially proposed that the differential sensitivity between the two
cell lines might be due to the different levels of Bcl-2 family death
proteins or survival proteins. However, using Western blotting with
densitometry, it was observed (Fig 1) that the total cellular expression of Bcl-2, Bcl-XL, and Bax
between the two cell lines was similar. The TNF-sensitive
CEM/VLB100 cell line did express higher levels of the death
proteins, Bcl-XS and Bad, compared with the parental CEM.
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| Fig 1.
The expression of Bcl-2 family proteins at the whole cell
level and subcellular distribution. Resting CEM and
CEM/VLB100 cells were dissolved with 1% Triton X-100
containing lysis buffer. Protein extract (50 µg/lane) was loaded on
to 12% SDS-PAGE, transferred to nitrocellulose membrane, and probed
with indicated anti-Bcl-2 family protein antibodies. -Actin served
as protein quality control with each experiment. The density of each
band was analyzed by densitometry. For whole cell lysate, the ratio
CEM:CEM/VLB100 for Bcl-XS = 29:71 (1:2.4);
ratio CEM:CEM/VLB100 for Bad = 38:62 (1:1.6). For
mitochondria protein, ratio CEM:CEM/VLB100 for
Bcl-XS = 3:97 (1:32); ratio CEM:CEM/VLB100
for Bad = 38:62 (1:1.6); ratio CEM:CEM/VLB100 for Bax = 12:88 (1:7.3). For the cytosol proteins, ratio
CEM:CEM/VLB100 for Bax = 62:38 (1.6:1).
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Subcellular distribution of Bcl-2 family proteins in the resting
state.
We were next interested in whether the distribution of Bcl-2 family
proteins in mitochondria was consistent with the whole cell levels.
Leukemic cells were broken with a homogenizer in the absence of
detergent. The postmitochondrial supernatant (or S10), which contains
light membrane (endoplasmic reticulum, nuclear envelope, and Golgi
complex), was removed by ultrafiltration. The subcellular distribution
of Bcl-2 family proteins, such as Bcl-2, Bcl-XL,
Bcl-XS, Bad, and Bax, were analyzed in both purified cytosol and mitochondria. Bcl-2 mainly located to mitochondria and was
not present in the cytosol as a soluble protein (Fig 1). However, Bcl-2
protein was also detected in the postmitochondrial supernatant
containing light membrane (results not shown). Bcl-XL distributed in both mitochondria as a membrane-bound protein and cytosol as a soluble protein. The expression of the antiapoptotic proteins, Bcl-2 and Bcl-XL, were similar between the two
cell lines in both cytosol and mitochondria. The proapoptotic proteins, Bcl-XS, Bad, and Bax, mainly located to the cytosol.
Interestingly, cells of the TNF--sensitive CEM/VLB100
cell line expressed more Bcl-XS, Bad, and Bax in their
mitochondria than the CEM cell line. The cytosolic expression of
Bcl-XS and Bad was similar between two cell lines. Thus, it
appears that the increased expression of Bcl-XS and Bad in
the CEM/VLB100 cell line at the whole cell level is due to
higher levels of these proteins in the mitochondria. Bax expression was
lower in the cytosol and higher in the mitochondria in the
CEM/VLB100 cell line compared with the CEM cell line. These results suggest that the ratio of death protein to survival protein at
the mitochondrial level is different from the whole cell level. The
increased susceptibility of CEM/VLB100 mitochondria to
TNF--induced respiratory inhibition30 may be partly due
to higher levels of these death proteins.
The redistribution of Bcl-2 proteins in response to
TNF- treatment.
We have suggested that redistribution of the Bcl-2 family proteins
might occur when leukemic cells are exposed to TNF-. Cells were
treated with 250 U/mL (12.5 ng/mL) TNF- for 3 hours. This concentration of TNF- has been reported to induce apoptosis in the
CEM/VLB100 cell line at 3 hours31 in the
absence of cell membrane leakage.30 Minimal apoptosis is
seen in the CEM cell line at this time point. The whole cell population
(including apoptotic and nonapoptotic cells) was obtained for the
preparation of cytosol and mitochondria. Redistribution of the
proapoptotic proteins, Bcl-XS, Bad, and Bax, from the
cytosol to mitochondria was clearly seen in both cell lines (Fig 2).
After exposure to TNF-, TNF-sensitive CEM/VLB100 cells
expressed more Bcl-XS, Bad, and Bax in their mitochondria
compared with CEM cells (Fig 2). TNF-
did not alter the distribution of Bcl-XL.
Bcl-XS translocation from cytosol to mitochondria was not
accompanied by Bcl-XL, suggesting that Bcl-XL
and Bcl-XS do not always share the same location. Increased
levels of Bcl-2 protein were seen in the mitochondria of the
TNF--resistant CEM cell line after TNF- treatment (control:TNF = 41:59). However, a reduction in Bcl-2 protein was seen in the TNF-sensitive CEM/VLB100 mitochondria (control:TNF = 54:46)
(Figs 2 and 3A). Using flow cytometry, we
found that Bcl-2, Bcl-X, Bad, and Bax were all detected on the
mitochondrial outer membrane (data not shown). We also confirmed that
Bcl-2 translocated to the mitochondrial outer membrane of CEM cells
after TNF- treatment as shown by flow cytometry. As described above,
there was no detectable soluble Bcl-2 protein in the cytosol of either
cell line. Using crude cytosol (S10), which contains light membrane, we
detected a reduction in Bcl-2 levels after exposure to TNF- (Fig
3B). This suggests that the increased Bcl-2 in the CEM mitochondria may
have moved from the light membrane, possibly from the rough endoplasmic
reticulum (RER). It is unclear why decreased Bcl-2 levels were seen in
both the light membrane and mitochondria of the CEM/VLB100
cell line during TNF--induced apoptosis.
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| Fig 2.
TNF--induced Bcl-2 family protein redistribution.
Cells were treated with 250 U/mL TNF- for 3 hours. Whole cell lysate
and cellular fractions were prepared, and the same amount of protein
(50 µg/lane) was analyzed by Western blotting. Ratios of
mitochondrial Bcl-2 family proteins after exposure to TNF- are
CEM:CEM/VLB100 for Bcl-XS = 33:67 (1:2);
CEM:CEM/VLB100 for Bad = 36:64 (1:1.8); and for Bax
= 12:88 (1:7.3). At the whole cell level, the CEM cell line the
ratio of control:TNF for Bcl-XS = 37:63 (1:1.7);
control:TNF for Bad = 29:71 (1:2.4); and the CEM/VLB100
cell line the ratio of control:TNF for Bcl-2 = 59:41
(1.4:1).
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| Fig 3.
TNF- induced Bcl-2 protein translocation from light
membrane to the mitochondrial outer membrane. CEM and
CEM/VLB100 cells were treated with 250 U/mL TNF- for 3 hours. (A) Freshly isolated mitochondria were blocked with BSA, labeled
with anti-Bcl-2 antibody, FITC-conjugated anti-mouse antibody, and
analyzed with flow cytometry. Control mouse IgG served as a negative
control. (B) Light membrane was dissolved with lysis buffer, and 50 µg protein was used for Western blotting. Protein concentration of
light membrane was confirmed by Bradford assay and by measuring optical
density 280 nm at 260 nm (RNA concentration). Ratio of Bcl-2,
control:TNF for CEM = 61:39 (1.6:1) for CEB/VLB100
=75:25 (3:1).
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We also examined whether TNF- induces changes in Bcl-2 family
proteins at the whole cell level. The expression of Bcl-XL and Bax at the whole cell level did not change after TNF- treatment (Fig 2). Increased expression of Bcl-XS and Bad was seen in
the CEM cell line after treatment with TNF-. However, a reduction in
Bcl-2 was observed in the CEM/VLB100 cell line after
TNF- treatment, as was also seen by the reductions in both
mitochondrial and light membrane levels. Although slight differences
were detected at the whole cell level with or without TNF-
treatment, such changes were not as great as those were at the
mitochondrial level.
TNF--induced cytochrome c release only
occurred in the CEM/VLB100 cell line.
TNF--induced targeting of BCL-2 family death proteins to
mitochondria was observed in both CEM and CEM/VLB100 cell
lines. We were therefore interested in whether death protein targeting was associated with cytochrome c release from mitochondria. We developed a triple staining method to identify cytochrome c
release. Mitochondria in the living cells were first stained with a red mitochondrion-selective vital dye, MitoTracker,33 and then
cytochrome c was stained green. MitoTracker staining has also
been used to assess m.34 Cytochrome c in
control cells localized to mitochondria, as red and green merged pixels
appeared orange/yellow (Fig 4A and D).
However, not all CEM/VLB100 cells took up MitoTracker, probably due to overexpression of the P-glycoprotein in the
cell membrane. Cells that did not take up MitoTracker displayed
punctuate green staining in their mitochondria (Fig 4D). After exposure to TNF- for 3 hours, no cytochrome c release was observed in the CEM cell line, as the cytochrome c distribution in
TNF-treated CEM cells was similar to that of untreated cells (Fig 4B).
By contrast, TNF-induced cytochrome c release was distinct in
the CEM/VLB100 cell line as shown by the green-labeled
cytochrome c image separating from the mitochondria with a
clearly diffuse pattern (Fig 4E). Nuclei were stained with DAPI and the
apoptotic cells were identified as cells containing condensed and
fragmented nuclei (Fig 4C and F). Mitochondria in the apoptotic cells
did not contain cytochrome c, as shown by the fact the
mitochondria were stained only with a red color (Fig 4E). Mitochondria
were well stained with MitoTracker in the apoptotic cells (Fig 4E), suggesting that TNF--induced m depolarization did not occur before nuclear fragmentation. Staining isolated mitochondria from TNF--treated cells with DiOC6(3), it was confirmed that
TNF--induced m reduction did not occur in either cell lines,
as mitochondria treated with or without TNF- showed similar response
to m depolarization induced by the uncoupler CCCP
(Fig 5). Enhanced MitoTracker dye uptake
was seen in the CEM/VLB100 cell line after TNF-
stimulation, possibly indicating inhibition of the P-glycoprotein
efflux pump due to decreased ATP supply by a dysfunctional
mitochondrial electron transport chain. These results indicate that CEM
mitochondria are more resistant to TNF--induced cytochrome
c release than those of the CEM/VLB100 cell line.
The differential sensitivity of the mitochondria in the two cell lines
to cytochrome c release may be due, in part, to the ratio of Bcl-2
family death proteins to survival proteins in both resting state and
following stimulation with apoptosis-inducing agents.
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| Fig 4.
TNF- induced cytochrome c release from
mitochondria. CEM cells (A through C) and CEM/VLB100 cells
(D through F) were incubated with or without 250 U/mL TNF- for 3 hours, labeled with MitoTracker, fixed, permeabilized, stained with
native anti-cytochrome c antibody, and finally counterstained
with DAPI as described in Materials and Methods. The stained cells were
examined by fluorescence microscopy. Cytochrome c antibody was
visualized with a fluorescent-conjugated anti-mouse IgG and assigned
the color green, whereas mitochondria labeled with MitoTracker were
assigned the color red. In the control cells (A and D) and CEM cells
treated with TNF- for 3 hours (B), red and green images were merged;
overlapping red and green pixels appears orange/yellow.
CEM/VLB100 cells treated with TNF- for 3 hours displayed
separation of the green (cytochrome c) from the mitochondria
(red) with appearance in the cytosol (E and F). C and F are cells
similar to B and E but counterstained with the nuclear dye DAPI. Cells
with condensed and/or fragmented nuclei are apoptotic cells
(arrows).
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| Fig 5.
TNF- did not induce  m reduction. After cells had
been treated with TNF- for 3 hours, isolated mitochondria were
incubated with or without 50 µmol/L uncoupler CCCP for 15 minutes at
37°C, followed by labeling with the m-sensitive dye
DiOC6(3).
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DISCUSSION |
In this study, we analyzed the subcellular distribution of Bcl-2 family
proteins in leukemic cells with differential sensitivity to apoptosis.
We proposed that the sensitivity of leukemic cells to TNF--induced
cytochrome c release and mitochondrial dysfunction may rely on
the ratio of Bcl-2 family death proteins to survival proteins at the
mitochondrial level. Despite extensive study of the relationship
between the expression of Bcl-2 family proteins and the sensitivity of
leukemic cells to apoptosis, controversy persists, and the importance
of the subcellular distribution of Bcl-2 family proteins has only
recently received attention. The observation that overexpression of
Bcl-2 and Bcl-XL in the mitochondrial outer membrane
prevents apoptosis by blocking cytochrome c release highlights
the importance of the mitochondrial location of the Bcl-2 family
proteins in the regulation of apoptosis.1-4
The CEM/VLB100 cell line is more susceptible to
TNF--induced apoptosis than the parent CEM cell
line.31,32 TNF- inhibits the mitochondrial electron
transport chain (ETC)30 and induces mitochondrial ultracondensation31 and cytochrome c
release from mitochondria to a greater extent in the
CEM/VLB100 cell line. We therefore proposed that the
increased susceptibility of the CEM/VLB100 cell line to
TNF- may be due to the characteristics of the mitochondria.
Expression of the antiapoptotic proteins, Bcl-2 and Bcl-XL,
was similar at the whole cell level in the two cell lines. Although an
increase in the expression of Bcl-XS and Bad was seen in
the CEM/VLB100 cell line, the differences were not
convincing enough to entirely account for the differences in the
susceptibility to apoptosis. In addition, the total protein contents of
Bcl-XS and Bad were much lower than other Bcl-2 family proteins.
The subcellular localization of Bcl-2, Bcl-XL, and Bax in
the CEM cell line was consistent with that described for HL-60 leukemia cells, murine thymocytes,11 and L929 murine fibrosarcoma
cells.12 Bcl-2 was exclusively membrane-bound, whereas Bax
was present predominantly in the cytosol and Bcl-XL was
present in both soluble and membrane-bound form. Bcl-XS and
Bad were mainly present in CEM cytosol as soluble proteins. There was
little Bcl-2 family death protein present in CEM mitochondria.
The Bcl-2 family death proteins, Bcl-XS, Bad, and Bax, were
present at greater levels in CEM/VLB100 mitochondria
compared with those of the CEM cell line. This increase in death
protein was not accompanied by an increase in the levels of survival
proteins. It has been reported that Bax binds to Bcl-2 via the BH3
domain to form a heterodimer, inactivating the normal ability of Bcl-2 to suppress apoptosis.21 Bad preferentially heterodimerizes with Bcl-XL through BH1 and BH2 interactions and functions
to antagonize the antiapoptotic properties of
Bcl-XL.28,29,36 However, the absence of BH1 and
BH2 regions in Bcl-XS suggests that Bcl-XS
enhances cellular sensitivity to apoptosis via a mechanism of action
distinct from other Bcl-2 family members that promote apoptosis,10 possibly due to blocking the binding of
cytochrome c to Bcl-XL.37 No difference
in Bcl-2 and Bcl-XL expression between the two cell lines
was observed at either the mitochondrial or the whole cell levels.
However, the higher levels of proapoptotic proteins in the mitochondria
may have diminished the antiapoptotic effect of both Bcl-2 and
Bcl-XL in the CEM/VLB100 cell line and therefore decrease the cellular apoptotic threshold. This may be one of
the reasons for the increased susceptibility of CEM/VLB100 cells to apoptotic stimuli.
It has been shown that Bax may translocate from the cytosol to the
mitochondria in leukemic cells during staurosporin-induced apoptosis.11,12 In addition, TNF- induces inhibition of
the mitochondrial ETC before other evidence of apoptosis in the
CEM/VLB100 cell line, suggesting involvement of the
mitochondria in the apoptotic process.30 We therefore
examined whether a proapoptotic stimuli, such as TNF-, induces
redistribution of Bcl-2 family proteins in the leukemic cells. We have
shown that the proapoptotic proteins, Bcl-XS, Bad, and Bax,
translocate from cytosol to mitochondria in both cell lines.
Translocation occurred to a similar degree, thus maintaining the higher
levels of the proapoptotic proteins in the CEM/VLB100 cell
line. The signal transduction pathway and mechanism, by which the
proapoptotic protein molecules transferred from soluble form to
membrane-bound proteins, remain unclear. However, these proapoptotic
proteins are not lethal to cells in the resting state.12
Bax and Bcl-XL do not form heterodimers in healthy
cells.38 Bad and Bax interfere with the antiapoptotic activity of Bcl-2 or Bcl-XL by binding to them and forming
nonfunctional heterodimers in challenged cells.29 The
translocation of proapoptotic proteins to the mitochondria may result
in the formation of channels or pores, which permit the release of
cytochrome c from within the mitochondria, a critical step in
the activation of the caspase protease cascade. The greater sensitivity
of the CEM/VLB100 mitochondria to TNF--induced
cytochrome c release and inhibition of the mitochondrial ETC
may be due to the higher levels of Bax, Bad, and Bcl-XS in the mitochondria and a resultant diminution of the antiapoptotic activity of Bcl-2 and Bcl-XL. We also observed that
TNF--induced mitochondrial inner membrane depolarization did not
occur before DNA fragmentation. This implies that the targeting of
proapoptotic proteins to mitochondria does not directly affect inner
membrane function. Consequently, TNF--induced inhibition of the
mitochondrial ETC may be the direct result of cytochrome c release.
Bcl-2 protein translocation from RER to mitochondria was observed in
the CEM cell line following exposure to TNF-. It is known that the
nuclear DNA-encoded mitochondrial membrane proteins are synthesized and
imported from the RER to the mitochondria. TNF- induces interaction
between the RER and mitochondria and increases autophagy in both CEM
and CEM/VLB100 cell lines.32 It is unknown
whether Bcl-2 translocation occurred to CEM/VLB100 mitochondria, because total cellular levels of Bcl-2 decreased in the
CEM/VLB100 cell line after exposure to TNF-. It has been found that Bcl-2 can be digested in vitro by active caspase-3 and
cleaved in vivo after activation of the Fas pathway in CEM cells.39 Therefore, it is possible that Bcl-2 may be
digested by caspase-3, which is only activated in the actively
apoptotic CEM/VLB100 cell line and not in CEM cells (Jia et
al, unpublished observations, July 1998). The cleavage of
Bcl-2, and subsequent failure to translocate to the mitochondria, may
further enhance cytochrome c release, permitting activation of
downstream caspases (such as caspase 3) and contribute to amplification
of the caspase cascade to ensure the inevitability of cell
death.39 This evidence suggests the existence of a feedback
loop between Bcl-2 and the caspases.
Thus, a number of mechanisms may operate to determine the sensitivity
of human leukemic cells to apoptotic stimuli. Apart from differing
cellular levels of proapoptotic or antiapoptotic proteins of the Bcl-2
family, these proteins may be favorably distributed to permit survival
of the cell or may translocate after toxic challenge to facilitate or
prevent death. BID, a member of the Bcl-2 family that directly mediates
cytochrome c release, has recently been shown to translocate to
the mitochondrial membrane,40,41 thus further supporting
the concept of intracellular movement of these proteins under
conditions of stress. We have described that the ratios of Bcl-2 family
death proteins to survival proteins at the whole cell level are
different to those in mitochondria. The susceptibility of leukemic
cells to TNF--induced cytochrome c release may rely
predominantly on the death proteins present in mitochondria.
Furthermore, cells reset their ratio of mitochondrial Bcl-2 proteins
depending on death or survival signaling.
| |
FOOTNOTES |
Submitted July 20, 1998; accepted November 19, 1998.
Supported by grant LRFG 9742 awarded by the Leukaemia Research Fund
(UK) to S.M.K. L.J. was supported as a post-doctoral research fellow by
this grant.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Stephen M. Kelsey, MD, Department of
Haematology, St Bartholomew's and the Royal London School of Medicine
and Dentistry, London E11BB, UK; e-mail: s.m.kelsey{at}mds.qmw.ac.uk.
| |
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ABSTRACT
INTRODUCTION