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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 5 (March 1), 1998:
pp. 1700-1705
Bcr-Abl Exerts Its Antiapoptotic Effect Against Diverse Apoptotic
Stimuli Through Blockage of Mitochondrial Release of Cytochrome C
and Activation of Caspase-3
By
Gustavo P. Amarante-Mendes,
Caryn Naekyung Kim,
Linda Liu,
Yue Huang,
Charles L. Perkins,
Douglas R. Green, and
Kapil Bhalla
From the Division of Hematology/Oncology, Department of Medicine,
Winship Cancer Center, Emory University School of Medicine, Atlanta,
GA; and the La Jolla Institute for Allergy & Immunology, San Diego, CA.
 |
ABSTRACT |
Bcr-Abl expression in leukemic cells is known to exert a potent
effect against apoptosis due to antileukemic drugs, but its mechanism
has not been elucidated. Recent reports have indicated that a variety
of apoptotic stimuli cause the preapoptotic mitochondrial release of
cytochrome c (cyt c) into cytosol, which mediates the cleavage and
activity of caspase-3 involved in the execution of apoptosis. Whether
Bcr-Abl exerts its antiapoptotic effect upstream to the cleavage and
activation of caspase-3 or acts downstream by blocking the ensuing
degradation of substrates resulting in apoptosis, has been the focus of
the present studies. In these, we used (1) the human acute myelogenous
leukemia (AML) HL-60 cells that are stably transfected with the
bcr-abl gene (HL-60/Bcr-Abl) and express p185 Bcr-Abl; and (2)
the chronic myelogenous leukemia (CML)-blast crisis K562
cells, which have endogenous expression of p210 Bcr-Abl. Exposure of
the control AML HL-60 cells to high-dose Ara-C (HIDAC),
etoposide, or sphingoid bases (including C2 ceramide, sphingosine, or sphinganine) caused the accumulation of cyt c in the
cytosol, loss of mitochondrial membrane potential (MMP), and increase
in the reactive oxygen species (ROS). These preapoptotic events were
associated with the cleavage and activity of caspase-3, resulting in
the degradation of poly (adenosine diphosphate
[ADP]-ribose) polymerase (PARP) and DNA fragmentation
factor (DFF), internucleosomal DNA fragmentation, and morphologic
features of apoptosis. In contrast, in HL-60/Bcr-Abl and K562 cells,
these apoptotic stimuli failed to cause the cytosolic accumulation of
cyt c and other associated mitochondrial perturbations, as well as the
failure to induce the activation of caspase-3 and apoptosis. While the
control HL-60 cells showed high levels of Bcl-2 and barely detectable
Bcl-xL, HL-60/Bcr-Abl cells expressed high levels of
Bcl-xL and undetectable levels of Bcl-2, a pattern of
expression similar to the one in K562 cells. Bax and caspase-3
expressions were not significantly different between HL-60/Bcr-Abl or
K562 versus HL-60 cells. These findings indicate that Bcr-Abl
expression blocks apoptosis due to diverse apoptotic stimuli upstream
by preventing the cytosolic accumulation of cyt c and other
preapoptotic mitochondrial perturbations, thereby inhibiting the
activation of caspase-3 and execution of apoptosis.
| |
INTRODUCTION |
CHRONIC MYELOGENOUS leukemia (CML) is a
malignancy of pluripotent hematopoietic cells caused by the
dysregulated activity of the tyrosine kinase (TK) encoded by the
chimeric bcr-abl gene.1 This fusion gene either
encodes for the p210 or p185 TK.1 These are implicated in
the pathogenesis of CML and approximately 25% of the adult
lymphoblastic leukemia (ALL), respectively.2
Bcr-Abl-expressing leukemic blasts are highly resistant to different
classes of chemotherapeutic drugs.3,4 Consistent with this,
cells derived from patients with CML in blast crisis (CML-BC), eg, K562
cells, which express p210 Bcr-Abl, have also been shown to be highly
resistant to antileukemic drug-induced apoptosis.5,6
Additionally, these cells are known to overexpress the antiapoptotic
Bcl-xL, but not Bcl-2.7 This may partly
contribute toward the resistance of these cells to high doses of
antileukemic drugs such as Ara-C or etoposide.6
Previous studies from our laboratory have shown that treatment of human
acute myelogenous leukemia (AML) HL-60 cells with high-dose Ara-C
(HIDAC) and etoposide causes cleavage and activity of caspase-3, which
results in the degradation of a number of substrates, including poly
(adenine diphosphate [ADP]-ribose) polymerase (PARP) and lamins,
producing the morphologic features of apoptosis.8,9
Caspase-3 activity has also been shown to cleave and activate a
recently cloned DNA fragmentation factor (DFF), which results in the
DNA fragmentation of apoptosis.10 More recent reports have
also shown that after treatment with antileukemic drugs, the cleavage
and activity of caspase-3 is promoted by the mitochondrial release and
accumulation of cyt c into the cytosol.11-13 This is
associated with the mitochondrial permeability transition, resulting in
the loss of membrane potential and increase in reactive oxygen species
(ROS).11,14 The three-dimensional structure of Bcl-2 and
Bcl-xL has similarity to the pore-forming domain of the
bacterial toxins, suggesting that Bcl-2 and Bcl-xL may be
channel proteins that regulate the transport of ions and small
proteins, like cyt c, across the outer mitochondrial
membrane.15 Overexpression of Bcl-2 or Bcl-xL
blocks the mitochondrial release of cyt c, thereby preventing the
activation of caspase-3 and apoptosis.11,13 Whether the
antiapoptotic effect of Bcr-Abl is due to the inhibition of the
upstream preapoptotic mitochondrial events or is exerted downstream
through the inhibition of the activity of caspase-3 has not been
elucidated. In the present studies using HL-60 cells with enforced
expression of p185 Bcr-Abl, as well as K562 cells, we show that Bcr-Abl
inhibits apoptosis due to diverse stimuli, including HIDAC, etoposide,
and sphingoid bases (ie, C2 ceramide, sphingosine, and
sphinganine) by blocking the mitochondrial release of cyt c and other
preapoptotic mitochondrial events, thus inhibiting the cleavage and
activity of caspase-3 and execution of apoptosis.
| |
MATERIALS AND METHODS |
Reagants.
Ara-C and etoposide were purchased from Sigma Chemicals (St Louis, MO).
Fas agonist CH11 (IgM) antibody was purchased from Kamiya Corp
(Seattle, WA). The tetrapeptide caspase inhibitor, zVAD-fmk, was
purchased from Bachem, Inc (Torrance, CA). C2 ceramide (N-acetyl sphingosine), D-erythrosphingosine (SO), and
d,l-erythro-dihydrosphingosine (Sphinganine, SA) were purchased from
Matreya, Inc (Pleasant Gap, PA).  -Interferon was purchased from
GIBCO (Grand Island, NY). Drugs were stored and reconstituted for
experiments as previously described.9 Rabbit anti-Bcl-x
and anti-Bax antisera and anti-Bcr-Abl antibody were purchased from
Pharmingen (San Diego, CA). Monoclonal anti-Bcl-2 (No. 124) was
obtained from DAKO Corp (Carpinteria, CA). Dr Ronald Jemmerson of the
University of Minnesota Medical School (Minneapolis, MN) kindly
provided the monoclonal antibody (MoAb) to cyt.12
Anti-caspase-3 and anti-Fas ligand (FasL) antibodies were purchased
from Transduction Laboratories (Lexington, KY). Anti-Fas antibody was
purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Rabbit
anti-DFF antisera was kindly provided by Dr Xiaodong Wang of the
University of Texas Southwestern School of Medicine (Dallas, TX).
Cells and transfection of the bcr-abl gene.
Human acute myeloid leukemia HL-60/Bcr-Abl and HL-60/neo cells were
created by transfection of the bcr-abl gene encoding the p185
Bcr-Abl and/or neomycin-resistant gene and passaged twice per
week, as previously described.16 K562 cells were passaged as previously reported.6 Logarithmically growing cells were used for the studies described below.
Western analyses of proteins.
Western analyses of Bcl-2, Bcl-xL, Bax, Bcr-Abl, DFF, and
 -actin were performed using specific antisera or MoAbs (see above), as previously described.17 Briefly, protein was extracted
from cells with lysis buffer (142.5 mmol/L KCl, 5 mmol/L
MgCl2, 10 mmol/L HEPES (pH 7.2), 1 mmol/L EGTA, 0.2% NP40,
and 0.2 mmol/L phenylmethylsulfonly fluoride [PMSF]) supplemented
with 0.2 trypsin inhibitory units/mL aprotinin, 0.7 µg/mL pepstatin,
and 1 µg/mL leupeptin. Appropriate protein amounts (20 µg) were
subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) (10% gel). After electrophoresis, proteins were transferred
to nitrocellulose sheets (0.5 A at 100 V; 4°C) for 1 to 3 hours. The blots were blocked in 5% nonfat dry milk solution for 3 hours at
room temperature with gentle shaking (5% nonfat milk
[wt/vol]/phosphate-buffered saline [PBS]/0.02% sodium azide [pH
7.4]). This was followed by incubation with the respective antibody
(1:1,000 dilution) at room temperature and then with antirabbit or
antimouse peroxidase-conjugated secondary IgG antibodies. Immune
complexes were detected with an enhanced chemiluminescence detection
method by immersing the blot for 1 minute in a 1:1 mixture of
chemiluminescence reagents A and B (Amersham UK, Little Chalfont, UK)
and then exposing to Kodak X-OMAT film (Eastman Kodak Co, Rochester,
NY) for a few seconds. Horizontal scanning densitometry was performed
on Western blots by using acquisition into Adobe Photo Shop (Apple,
Inc, Cupertino, CA) and analysis by the NIH Image Program (US National Institutes of Health, Bethesda, MD).
Preparation of S-100 fraction and Western analysis for cytochrome c.
Untreated and drug-treated cells were harvested by centrifugation at
1,000g for 10 minutes at 4°C. The cell pellets were washed once with ice-cold PBS and resuspended with 5 vol of buffer (20 mmol/L
HEPES-KOH, pH 7.5, 10 mmol/L KCl, 1.5 mmol/L KCl, 1.5 mmol/L MgCl2, 1 mmol/L sodium EDTA, 1 mmol/L sodium EGTA, 1 mmol/L
dithiothreitol, and 0.1 mmol/L PMSF), containing 250 mmol/L sucrose.
The cells were homogenized with a 22-gauge needle, and the homogenates
were centrifuged at 100,000g for 15 minutes at 4°C (S-100
fraction). The supernatants were collected and the protein
concentrations of S-100 were determined by Bradford method (Bio-Rad,
Hercules, CA). A total of 20 to 30 µg of S-100 was used
for Western blot analysis of cyt c, as described
previously.9,17
Measurement of mitochondrial potential and ROS.
In the untreated and drug-treated HL-60/neo, HL-60/Bcr-Abl, and K562
cells, to assess the changes in mitochondrial potential and ROS, 5 × 105 cells were incubated for 15 minutes at 37°C
with 40 nmol/L 3,3 dihexyloxacarbocyanine iodide
(DiOC6[3]) and 5 µmol/L
dichlorodihydrofluorescein diacetate (DCFH-DA), respectively and
analyzed by fluorescence-activated cell sorting (FACS) as described
previously.11,14,18
In vitro PARP cleavage activity of caspase-3.
In vitro translated, 35S-labeled PARP was prepared as
described previously.19 The caspase activity of caspase-3
was determined by its ability to degrade the in vitro-translated,
35S-labeled PARP into its 85- and 31-kD fragments, as
described previously.9,11
Detection of internucleosomal fragmentation of genomic DNA by
agarose gel electrophoresis.
After incubations with the designated concentrations and schedules of
the drugs, 1 × 106 cells were pelleted. The genomic
DNA was extracted and purified, and its purity was determined
spectrophotometrically.20 Agarose gel electrophoresis of
1.0 µg of DNA was performed as described previously.20
Morphology of apoptotic cells.
After treatment with or without drug, 50 × 103 cells
were washed with PBS (pH 7.3) and resuspended in the same buffer.
Cytospin preparations of the cell suspensions were fixed and stained
with Wright stain. Cell morphology was determined by light microscopy. In all, five different fields were randomly selected for counting of at
least 500 cells. The percentage of apoptotic cells was calculated for
each experiment, as previously described.20
| |
RESULTS |
Effect of HIDAC or etoposide on Bcr-Abl expression, cytosolic
accumulation of cyt c, caspase-3 activity and apoptosis.
The effects of HIDAC and etoposide treatment on the mitochondrial
release of cyt c, with resultant activation of the caspase-3 activity,
DNA fragmentation, and the morphologic features of apoptosis were
compared in HL-60/Bcr-Abl and K562 cells versus the control HL-60/neo
cells. Figure 1A shows that following an
exposure to 100 µmol/L Ara-C for 4 hours, a marked increase in the
cytosolic cyt c level was observed in HL-60/neo cells, but not in
either the HL-60/Bcr-Abl or K562 cells. Although not shown here, after exposure to apoptotic stimuli, the cytosolic accumulation of the cyt c
is due to its release from the mitochondria.11,13 The ability of HIDAC-mediated accumulation of cytosolic cyt c to trigger the protease activity of caspase-3 was determined by examining (1)
whether the S-100 fraction from the untreated or HIDAC-treated cells
would promote the degradation of the in vitro-translated PARP into its
85-kD and 31-kD cleaved products; or (2) whether the S-100 fraction
would show the cleavage of the 45-kD subunit of DFF into 30-kD and,
later, completely into 11-kD fragments.10 Figure 1B and C
show that the S-100 fraction of only the HIDAC-treated HL-60/neo cells
produced the degradation of the in vitro-translated PARP (Fig 1B) and
showed the complete cleavage of the 45-kD subunit of DFF into its 11-kD
fragment (Fig 1C), which is known to promote the DNA fragmentation of
apoptosis.10 Neither the PARP nor DFF cleavage was observed
in S-100 fractions of the HIDAC-treated HL-60/Bcr-Abl or K562 cells or
of the untreated three cell-types (Fig 1B and C). Correspondingly,
HIDAC treatment caused internucleosomal DNA fragmentation in HL-60/neo
cells (Fig 1D, lane 2), but not in HL-60/Bcr-Abl and K562 cells (Fig
1D, lanes 4 and 6). Lanes 1, 3, and 5 contain DNA from untreated
HL-60/neo, HL-60/Bcr-Abl, and K562 cells, respectively. Figure 1E and F
show that after the exposure to 50 µmol/L etoposide for 4 hours, only
the HL-60/neo cells showed the cytosolic accumulation of cyt c and the
degradation of the in vitro-translated PARP into its cleaved products.
Again, this was not observed in the etoposide-treated HL-60/Bcr-Abl
(Fig 1E and F) or K562 cells (not shown). The S-100 fractions of
etoposide-treated HL-60/neo cells also showed the cleavage of the 45-kD
DFF subunit into its 11-kD fragment, which was not observed in
HL-60/Bcr-Abl or K562 cells (data not shown). The concentrations of
Ara-C and etoposide selected for these experiments have been previously shown to be potent inducers of DNA fragmentation and the morphologic features of apoptosis in the control HL-60 cells.6,8,11,20 The results presented in Fig 1A through F are representative of three
separate experiments.
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| Fig 1.
Western analysis of cytosolic levels of cyt c (A); 116-kD
35S-labeled, in vitro-translated PARP or its cleaved
products (B); 45-kD DFF subunit or its 11-kD cleavage fragment (C); as
well as the internucleosomal DNA fragmentation in the untreated and HIDAC-treated HL-60/neo (D, lanes 1 and 2), HL-60/Bcr-Abl (lanes 3 and
4), or K562 cells (lanes 5 and 6). Lane M in (D) represents the 123-bp
marker DNA ladder. (E and F) Show Western analyses of the levels of
cytosolic cyt c (E) and PARP or its cleaved products (F) in the
untreated or etoposide-treated HL-60/neo or HL-60/Bcr-Abl cells.
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Loss of mitochondrial membrane potential (MMP) and increase in ROS
during apoptosis.
We have recently reported that HIDAC- or etoposide-induced cytosolic
accumulation of cyt c is followed by a decrease in the inner MMP and an
increase in the ROS, while these preapoptotic mitochondrial
perturbations are blocked by overexpression of Bcl-xL and
Bcl-2.11,13 Figure 2 shows the
effect of HIDAC treatment (100 µmol/L for 4 hours) on the percentage
of HL-60/neo, HL-60/Bcr-Abl, or K562 cells that displayed either low
MMP (  M) or increased ROS, as detected by the cellular uptake of
the fluorochrome, DiOC6 (for MMP), or the staining by
DCFH-DA (for ROS), respectively. As shown, HIDAC treatment caused a
significant increase in the percentage of cells (39%; control, < 3.5%) showing low MMP and significantly higher levels of ROS ( 33%;
control, < 2.0%) in the HL-60/neo cells, but not in the
HL-60/Bcr-Abl or K562 cell populations (P < .001, paired
t-test). Similar results were obtained in etoposide-treated
HL-60/neo versus HL-60/Bcr-Abl or K562 cells (data not shown). These
findings are representative of three separate experiments.
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| Fig 2.
Reduction of the inner mitochondrial membrane potential
( M, A) and production of ROS (B) in untreated control or
Ara-C-treated (4 hours) HL-60/neo, HL-60/Bcr-Abl, and K562 cells.
Ara-C treatment increased the percentage of HL-60/neo, but not
HL-60/Bcr-Abl or K562 cells, which displayed either low M
(DiOC6[3] uptake) or high ROS production (staining by
DCFH-DA). The open peak (indicated by the arrow) represents the
positive control after treatment with 10 mmol/L
H2O2.
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Expression of Bcr-Abl, caspase-3, Bcl-xL, Bcl-2, and Bax.
The levels of Bcr-Abl, Abl, caspase-3, Bcl-xL, Bcl-2, and
Bax were determined by Western analyses of the protein extracts of
untreated or HIDAC-treated HL-60/neo, HL-60/Bcr-Abl, and K562 cells.
Figure 3A shows that HL-60/Bcr-Abl and
K562, but not HL-60/neo, cells express p185 and p210 Bcr-Abl,
respectively. In contrast, all of the cell-types express p145 Abl.
Following Ara-C treatment for 4 hours, neither Bcr-Abl nor Abl levels
were altered in any of the cell-types (Fig 3A). In contrast, exposure
to Ara-C resulted in a significant decline in the level of 32-kD
caspase-3 levels due to its cleavage and activation in HL-60/neo, but
not in HL-60/Bcr-Abl or K562 cells (Fig 3B). It is noteworthy that
although the latter two cell types are resistant to apoptosis, their
32-kD caspase-3 levels are not significantly different from those in
HL-60/neo cells (Fig 3B), when normalized for the levels of actin
serving as the control. We have previously reported that HIDAC-induced downregulation of the 32-kD caspase-3 levels in HL-60/neo cells is due
to its cleavage into 20- and 12-kD fragments.8 As
previously noted for HL-60 cells, Fig 3C and D show that HL-60/neo
cells have high Bcl-2, but barely detectable Bcl-xL
levels.6 Confirming our previous findings,38
the enforced expression of p185 Bcr-Abl in HL-60 cells resulted in a
dramatic downregulation of Bcl-2 and an associated upregulation of
Bcl-xL. It is noteworthy that a similar pattern of
expression of these proteins has been observed in K562
cells.6 There was no significant difference in Bax levels
among the various cell types, when normalized to the intracellular actin levels serving as the control. It is also noteworthy that, except
for the caspase-3 levels, HIDAC treatment did not affect the levels of
these proteins in any of the cell types (Fig 3A, C, D, and E).
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| Fig 3.
Western analyses of Bcr-Abl, Abl, 32 kD caspase-3,
Bcl-xL, Bcl-2, and Bax levels in the untreated or
Ara-C-treated HL-60/neo, HL-60/Bcr-Abl or K562 cells.
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Cytosolic cyt c accumulation and caspase-3 activity caused by
sphingolipids.
We next determined whether apoptotic stimuli other than the
antileukemic drugs Ara-C and etoposide cause cyt c accumulation in the
cytosol and trigger caspase-3 activity in HL-60/neo cells, as well as
whether these are affected by the expression of Bcr-Abl in
HL-60/Bcr-Abl or K562 cells. Exposure of HL-60/neo cells to the
sphingoid bases C2 ceramide (50 µmol/L), sphingosine (10 µmol/L), or sphinganine (10 µmol/L) for 6 hours, which have been
previously shown to induce apoptosis in HL-60
cells,21 clearly caused significant cytosolic
accumulation of cyt c (Fig 4A) and the
generation of the in vitro-translated PARP cleavage activity of
caspase-3 (Fig 4B). These molecular events were blocked in
HL-60/Bcr-Abl and K562 cells. Figure 4C shows that in HL-60/neo cells
cotreatment with zVAD-fmk, a tetrapeptide inhibitor of caspases
including caspase-3,22 did not affect the cytosolic
accumulation of cyt c due to C2 ceramide, sphingosine (Fig
4, lanes 5 and 6), and sphinganine (not shown); but prevented the PARP
cleavage activity of caspase-3 and the DNA fragmentation of apoptosis
due to these sphingoid bases (data not shown).
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| Fig 4.
Western analyses of the levels of cytosolic cyt c (A) and
116 kD 35S-labeled, in vitro-translated PARP or its cleaved
products (B) in untreated and sphingosine (SO), sphinganine (SA), or
C2 ceramide-treated HL-60/neo, HL-60/Bcr-Abl, and K562
cells. (C) Shows Western analysis of the cytosolic cyt c levels in
HL-60/neo cells after exposure to SO, C2 ceramide, or zVAD
alone; or zVAD plus SO or C2 ceramide (C) (see text).
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DISCUSSION |
Although previous reports have shown that Bcr-Abl expression confers
resistance against the antileukemic drug-induced DNA fragmentation and
morphologic features of apoptosis, the mechanism or the step at which
this antiapoptotic effect is exerted has not been clearly
defined.23 Results of present studies using the human AML
HL-60 cells have highlighted several novel findings. We show for the
first time that (1) treatment with the antileukemia drugs, Ara-C or
etoposide, as well as exposure to sphingoid bases (C2
ceramide, sphingosine, or sphinganine) cause the mitochondrial release
and cytosolic accumulation of cyt c; (2) Ara-C or etoposide treatment
also induces the cleavage and activity of DFF, which promotes the DNA
fragmentation of apoptosis; and (3) Bcr-Abl expression results in the
inhibition of the preapoptotic mitochondrial perturbations, thereby
blocking the generation of caspase activity and apoptosis.
The precise mechanism by which apoptotic stimuli cause the egress of
cyt c from the inner mitochondrial membrane to the cytosol, followed by
the loss of MMP and increase in ROS, has not been elucidated.24 The mechanism by which the cytosolic cyt c
brings about the cleavage and activation of caspase-3 in the presence of deoxy adenosine triphosphate (dATP) (or deoxy ADP [dADP]) and two
additional apoptotic protease activation factors (APAF-1 and APAF-3)
has also not been determined.12 More recently, however, a
clearer picture of this has emerged. Human APAF-1 has been shown to
have nucleotide binding sites and to bind cyt c. APAF-1 also possesses
in its N terminus domains sequence homology to the proapoptotic ced
4 and ced 3 genes of Caenorhabditis elegans; the
latter gene is homologous to caspase-3.25 Nonetheless, it
remains unclear how the TK encoded by Bcr-Abl either blocks the
stimulus for the release of cyt c from mitochondria or prevents its
accumulation in the cytosol. Phosphorylation of Bcl-2 and
Bcl-xL has also been shown to affect their antiapoptotic
effects, perhaps through an alteration of their function as the channel
proteins residing in the outer mitochondrial membrane and regulating
the trafficking of ions and small molecular weight proteins such as cyt
c.15,23,26-30 But, there is no evidence that Bcr-Abl causes
a posttranslational modification of Bcl-2, Bcl-xL, or Bax
by affecting their phosphorylation status (Fig 3). Phosphorylation of
Bad, a proapoptotic member that heterodimerizes with Bcl-xL
or Bcl-2, has been shown to be a potential mechanism through which
interleukin (IL)-3 mediates its antiapoptotic effect on the bone marrow
progenitor cells.31,32 Whether Bcr-Abl initiates a cascade
of events that results in phosphorylation of Bad is currently under
investigation. It seems unlikely that Bcr-Abl would directly
phosphorylate Bad, as this was shown to occur on the serine residues.
However, it is possible that a serine/threonine kinase such as Raf-1
could be activated by Bcr-Abl and be responsible for at least part of
its antiapoptotic effect. The ectopic expression of p210 Bcr-Abl into
MO7, 32Dc13, and FDC-P1 cells has been shown to result in a
constitutively hyperphosphorylated and activated Raf-1.33
Raf-1 is known to phosphorylate Bad, although on a different residue
from the one phosphorylated by IL-3-initiated signalling
pathway.34 A previous report had indicated that in the
mouse hematopoietic Ba/F3 cells, Bcr-Abl expression induces
Bcl-2 mRNA expression, thereby suppressing apoptosis of these cells
resulting from IL-3 withdrawal.35 Again, this is not the
case in the Bcr-Abl-expressing human HL-60/Bcr-Abl or K562 cells. In
HL-60/Bcr-Abl cells, Bcl-2 protein levels are almost completely
downregulated by the enforced expression of Bcr-Abl, while in K562
cells, the endogenous expression of Bcr-Abl is associated with barely
detectable levels of Bcl-2 (Ray et al6 and Fig 3). However,
both in HL-60Bcr-Abl and K562 cells, Bcl-xL is upregulated
(Fig 3). Because marked suppression of Bcl-2, which like
Bcl-xL has also been shown to inhibit the mitochondrial
release of cyt c, occurred concomitantly with Bcr-Abl-mediated
induction of Bcl-xL, the latter is unlikely to be the sole
explanation for the inhibition of cytosolic accumulation of cyt c due
to various apoptotic stimuli. This is also supported by our previously
reported observation that the enforced expression of the proapoptotic
Bcl-xS in K562 cells, which cancels the antiapoptotic
activity of Bcl-xL, only partially sensitizes K562 cells to
drug-induced apoptosis.6 Also, other cell types that
possess high intracellular levels of both Bcl-xL and Bcl-2
still retain the ability to undergo apoptosis after exposure to
chemotherapeutic drugs.36 Taken together, the alterations
in the levels of Bcl-2 and Bcl-xL observed in association
with Bcr-Abl expression do not appear to be the sole mechanisms
underlying the blockage of the preapoptotic mitochondrial perturbations
observed in the HL-60/Bcr-Abl and K562 cells.
A previous report had indicated that Bcr-Abl expression in myeloid
leukemia cells prevents Fas-induced apoptosis,37,38 but
does not prevent apoptotic death mediated by cytotoxic T cells, human
natural killer, or lymphokine-activated cells.39,40
Recently, we have confirmed that Bcr-Abl expression confers resistance
against Fas-induced apoptosis (data not shown). Treatment of HL-60
cells by -interferon (400 U for 48 hours) followed by CH11 anti-Fas agonist antibody (1 µg/mL for 24 hours), which triggers FasR-mediated signalling, caused the cytosolic accumulation of cyt c and induced the
PARP cleavage activity of caspase-3. These cytosolic events were
blocked in HL-60/Bcr-Abl cells (data not shown). During apoptosis due
to treatment with sphingoid bases or the triggering of Fas death-receptor signalling, the direct cleavage and activation of
caspase-3 had been previously reported.41-43 Our findings
highlight the contribution of the cytosolic accumulation of cyt c due
to these apoptotic stimuli in promoting the cleavage and activity of
caspase-3. These data are also consistent with the previous observations showing that Bcl-2 or Bcl-xL overexpression
inhibits cytosolic accumulation of cyt c, thereby inhibiting apoptosis due to Fas signalling or sphingoid bases.44-46 In this
context, the upstream inhibitory effect of Bcr-Abl on the molecular
cascade of apoptosis indicates an additional mechanism of resistance to sphingoid bases or Fas-induced apoptosis.47
| |
FOOTNOTES |
Submitted August 11, 1997;
accepted October 17, 1997.
G.P.A.-M. has been a Brazilian Research Council (CNPq) Fellow.
Address reprint requests to Kapil Bhalla, MD, Division of
Hematology/Oncology, Emory University School of Medicine, Winship Cancer Center, 1365-B Clifton Rd, NE, Atlanta, GA 30322.
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.
| |
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March 1, 2001;
59(3):
453 - 461.
[Abstract]
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R. Nimmanapalli, M. Porosnicu, D. Nguyen, E. Worthington, E. O’Bryan, C. Perkins, and K. Bhalla
Cotreatment with STI-571 Enhances Tumor Necrosis Factor {{alpha}}-related Apoptosis-inducing Ligand (TRAIL or Apo-2L)- induced Apoptosis of Bcr-Abl-positive Human Acute Leukemia Cells
Clin. Cancer Res.,
February 1, 2001;
7(2):
350 - 357.
[Abstract]
[Full Text]
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M. M. Mc Gee, G. Campiani, A. Ramunno, C. Fattorusso, V. Nacci, M. Lawler, D. C. Williams, and D. M. Zisterer
Pyrrolo-1,5-benzoxazepines Induce Apoptosis in Chronic Myelogenous Leukemia (CML) Cells by Bypassing the Apoptotic Suppressor Bcr-Abl
J. Pharmacol. Exp. Ther.,
January 1, 2001;
296(1):
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[Abstract]
[Full Text]
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M. W. N. Deininger, J. M. Goldman, and J. V. Melo
The molecular biology of chronic myeloid leukemia
Blood,
November 15, 2000;
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3343 - 3356.
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G. Fang, C. N. Kim, C. L. Perkins, N. Ramadevi, E. Winton, S. Wittmann, and K. N. Bhalla
CGP57148B (STI-571) induces differentiation and apoptosis and sensitizes Bcr-Abl-positive human leukemia cells to apoptosis due to antileukemic drugs
Blood,
September 15, 2000;
96(6):
2246 - 2253.
[Abstract]
[Full Text]
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Y. Sonoda, Y. Matsumoto, M. Funakoshi, D. Yamamoto, S. K. Hanks, and T. Kasahara
Anti-apoptotic Role of Focal Adhesion Kinase (FAK). INDUCTION OF INHIBITOR-OF-APOPTOSIS PROTEINS AND APOPTOSIS SUPPRESSION BY THE OVEREXPRESSION OF FAK IN A HUMAN LEUKEMIC CELL LINE, HL-60
J. Biol. Chem.,
May 19, 2000;
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[Abstract]
[Full Text]
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C. Oetzel, T. Jonuleit, A. Götz, H. van der Kuip, H. Michels, J. Duyster, M. Hallek, and W. E. Aulitzky
The Tyrosine Kinase Inhibitor CGP 57148 (ST1 571) Induces Apoptosis in BCR-ABL-positive Cells by Down-Regulating BCL-X
Clin. Cancer Res.,
May 1, 2000;
6(5):
1958 - 1968.
[Abstract]
[Full Text]
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M. Horita, E. J. Andreu, A. Benito, C. Arbona, C. Sanz, I. Benet, F. Prosper, and J. L. Fernandez-Luna
Blockade of the Bcr-Abl Kinase Activity Induces Apoptosis of Chronic Myelogenous Leukemia Cells by Suppressing Signal Transducer and Activator of Transcription 5-dependent Expression of Bcl-xL
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March 13, 2000;
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[Abstract]
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C. L. Perkins, G. Fang, C. N. Kim, and K. N. Bhalla
The Role of Apaf-1, Caspase-9, and Bid Proteins in Etoposide- or Paclitaxel-induced Mitochondrial Events during Apoptosis
Cancer Res.,
March 1, 2000;
60(6):
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[Abstract]
[Full Text]
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P. le Coutre, E. Tassi, M. Varella-Garcia, R. Barni, L. Mologni, G. Cabrita, E. Marchesi, R. Supino, and C. Gambacorti-Passerini
Induction of resistance to the Abelson inhibitor STI571 in human leukemic cells through gene amplification
Blood,
March 1, 2000;
95(5):
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[Abstract]
[Full Text]
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C. Perkins, C. N. Kim, G. Fang, and K. N. Bhalla
Arsenic induces apoptosis of multidrug-resistant human myeloid leukemia cells that express Bcr-Abl or overexpress MDR, MRP, Bcl-2, or Bcl-xL
Blood,
February 1, 2000;
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1014 - 1022.
[Abstract]
[Full Text]
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P. A. Svingen, A. Tefferi, T. J. Kottke, G. Kaur, V. L. Narayanan, E. A. Sausville, and S. H. Kaufmann
Effects of the bcr/abl Kinase Inhibitors AG957 and NSC 680410 on Chronic Myelogenous Leukemia Cells in Vitro
Clin. Cancer Res.,
January 1, 2000;
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[Abstract]
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C. Scaffidi, I. Schmitz, J. Zha, S. J. Korsmeyer, P. H. Krammer, and M. E. Peter
Differential Modulation of Apoptosis Sensitivity in CD95 Type I and Type II Cells
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August 6, 1999;
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[Abstract]
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R. G. Wickremasinghe and A. V. Hoffbrand
Biochemical and Genetic Control of Apoptosis: Relevance to Normal Hematopoiesis and Hematological Malignancies
Blood,
June 1, 1999;
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Q. P. Dou, T. F. McGuire, Y. Peng, and B. An
Proteasome Inhibition Leads to Significant Reduction of Bcr-Abl Expression and Subsequent Induction of Apoptosis in K562 Human Chronic Myelogenous Leukemia Cells
J. Pharmacol. Exp. Ther.,
May 1, 1999;
289(2):
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[Abstract]
[Full Text]
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P. Ghafourifar, S. D. Klein, O. Schucht, U. Schenk, M. Pruschy, S. Rocha, and C. Richter
Ceramide Induces Cytochrome c Release from Isolated Mitochondria. IMPORTANCE OF MITOCHONDRIAL REDOX STATE
J. Biol. Chem.,
March 5, 1999;
274(10):
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[Abstract]
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N. Takeda, M. Shibuya, and Y. Maru
The BCR-ABL oncoprotein potentially interacts with the xeroderma pigmentosum group B protein
PNAS,
January 5, 1999;
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[Abstract]
[Full Text]
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Q. Chen, N. Takeyama, G. Brady, A. J.M. Watson, and C. Dive
Blood Cells With Reduced Mitochondrial Membrane Potential and Cytosolic Cytochrome C Can Survive and Maintain Clonogenicity Given Appropriate Signals to Suppress Apoptosis
Blood,
December 15, 1998;
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[Abstract]
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L. M. Martins, T. J. Kottke, S. H. Kaufmann, and W. C. Earnshaw
Phosphorylated Forms of Activated Caspases Are Present in Cytosol From HL-60 Cells During Etoposide-Induced Apoptosis
Blood,
November 1, 1998;
92(9):
3042 - 3049.
[Abstract]
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G. S. Burgess, E. A. Williamson, L. D. Cripe, S. Litz-Jackson, J. A. Bhatt, K. Stanley, M. J. Stewart, A. S. Kraft, H. Nakshatri, and H. S. Boswell
Regulation of the c-jun Gene in p210 BCR-ABL Transformed Cells Corresponds With Activity of JNK, the c-jun N-Terminal Kinase
Blood,
October 1, 1998;
92(7):
2450 - 2460.
[Abstract]
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B.-J. Kroesen, B. Pettus, C. Luberto, M. Busman, H. Sietsma, L. de Leij, and Y. A. Hannun
Induction of Apoptosis through B-cell Receptor Cross-linking Occurs via de Novo Generated C16-Ceramide and Involves Mitochondria
J. Biol. Chem.,
April 20, 2001;
276(17):
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[Abstract]
[Full Text]
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A. H. Boulares, A. J. Zoltoski, A. Yakovlev, M. Xu, and M. E. Smulson
Roles of DNA Fragmentation Factor and Poly(ADP-ribose) Polymerase in an Amplification Phase of Tumor Necrosis Factor-induced Apoptosis
J. Biol. Chem.,
October 5, 2001;
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[Abstract]
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Abstract
Introduction
Abstract