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Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2415-2422
BCR-ABL Delays Apoptosis Upstream of Procaspase-3 Activation
By
Laurence Dubrez,
Béatrice Eymin,
Olivier Sordet,
Nathalie Droin,
Ali G. Turhan, and
Eric Solary
From the Department of Biology and Therapy of Cancer, INSERM CJF
94-08, Faculty of Medicine, Dijon, and INSERM U 362, IGR, Villejuif,
France.
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ABSTRACT |
The p210bcr-abl protein was shown to inhibit apoptosis
induced by DNA damaging agents. Apoptotic DNA fragmentation is delayed
in the bcr-abl+ K562 and KCL-22 compared with the
bcr-abl U937 and HL-60 cell lines when treated
with etoposide concentrations that induce similar DNA damage in the
four cell lines. By the use of a cell-free system, we show that nuclei
from untreated cells that express p210bcr-abl remain
sensitive to apoptotic DNA fragmentation induced by triton-soluble extracts from p210bcr-abl cells treated with etoposide.
In the four tested cell lines, apoptotic DNA fragmentation is
associated with a decreased expression of procaspase-3
(CPP32/Yama/apopain) and its cleavage into a p17 active fragment,
whereas the long isoform of procaspase-2 (ICH-1L) remains unchanged and
the poly(adenosine diphosphate-ribose)polymerase protein is cleaved.
These events are delayed in bcr-abl+ compared
with bcr-abl cell lines. The role of
p210bcr-abl in this delay is confirmed by comparing the
effect of etoposide on the granulocyte-macrophage colony-stimulating
factor (GM-CSF)-dependent UT7 cells and the
bcr-abl-transfected GM-CSF-independent UT7/9 clone. We
conclude that the cytosolic pathway that leads to apoptotic DNA
fragmentation in etoposide-treated leukemic cells is delayed upstream
of procaspase-3-mediated events in bcr-abl+ cell
lines.
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INTRODUCTION |
THE PHILADELPHIA chromosome is the
cytogenetic hallmark of chronic myelogenous leukemia
(CML).1 Translocation of the c-abl gene, located on
chromosome 9q, within the bcr gene, located on chromosome 22q,
results in the formation of a chimeric protein, p210bcr-abl.2 This event is generally accepted
to be the primary initiating event in the genesis of CML because mice
transplanted with bone marrow cells infected with retroviral vectors
encoding p210bcr-abl develop a CML-like
disease.3 The c-ABL protein is a nuclear protein with low
intrinsic tyrosine kinase activity, whereas the BCR-ABL protein is a
cytoplasmic, membrane-associated protein that contains a constitutively
high level of tyrosine kinase activity. It was initially assumed that
CML was caused by uncontrolled cell proliferation resulting in the
clonal expansion evident in this disease.4 Actually, CML
progenitors have similar proliferation rates as their normal
counterparts.5 Recent studies have shown that both the
v-abl transforming oncogene product and the
p210bcr-abl prolonged hematopoietic cell survival by
inhibition of apoptosis.6-8 The BCR-ABL chimeric protein
confers on hematopoietic cells the ability to survive treatments such
as growth factor deprivation, cytotoxic drugs, protein tyrosine kinase
inhibitors, and Fas-ligand.9-12 Antisense oligonucleotides
that downregulate BCR-ABL protein expression in CML cell lines either
induce apoptotic cell death13 or render the cells more
susceptible to cell death induction by cytotoxic drugs and
Fas-ligand.9,10 The mechanisms by which the deregulated v-ABL and BCR-ABL tyrosine kinases delay apoptotic cell death remain
poorly understood. Translocation of the protein kinase C
 II,14 increased glucose uptake,15 delayed
G2/M transition after DNA damage,9 and activation of Ras
functions16 were proposed to mediate this inhibitory
effect, whereas the MAP kinase kinase/MAP kinase pathway
was shown not to be required for BCR-ABL-mediated suppression of
apoptosis.17
Transduction of the apoptotic signal and execution of apoptosis require
the coordinate action of several aspartate-specific cysteine proteases,
the so-called caspases. The 10 human caspases identified so far can be
divided into four subfamilies based on their structure and their
homology to the human prototype interleukin-1 converting enzyme
(ICE) and the nematode prototype CED-3.18 These subfamilies
are the ICE-like caspases, the CED-3-like caspases, the caspases that
contain prodomains highly related to the death effector domain of the
Fas/APO-1 receptor, and the NEDD2/ICH-1 caspases. All these enzymes are
initially synthesized as single-chain inactive proenzymes that require
cleavage after aspartate residues to obtain the active protease. Under
physiological conditions, this process probably involves heterotypic
protein-protein interactions and a cascade of caspases.19
Although molecular ordering of this cascade is only partly known, it
seems that several apoptotic pathways probably exist. For example,
deletion of the ICE (caspase-1) gene inhibits Fas-induced apoptosis
without modifying several other apoptotic pathways.20 By
contrast, activation of CED-3-like caspases could be common to most
apoptotic pathways.21 Distinct from ICE, these caspases
cleave the cell death substrate poly(adenosine diphosphate
[ADP]-ribose) polymerase (PARP) into signature apoptotic fragments.22-24 Activation of CED-3-related caspases was
shown recently to be controlled by the CED-9/Bcl-2 family of molecules. This control was suggested to take place at the mitochondrial membrane25 and to involve the so-far unidentified mammalian homolog of the nematode protein CED-4.26
In the present study, we addressed the question of the relationship
between BCR-ABL-mediated inhibition of apoptosis and the activation of
CED-3-related caspases. As a model system, we used etoposide
(VP-16)-induced apoptosis. This topoisomerase II-reactive agent
produces double-strand breaks by stabilizing a transient intermediate
of the topoisomerase reaction.27 We have shown previously
that etoposide induced similar levels of these DNA damage in
bcr-abl+ (K562, KCL-22) and
bcr-abl (HL-60, U937) cell lines.28
However, apoptosis was strongly delayed in bcr-abl+
cells. Here, we show that this delay occurs upstream of procaspase-3 activation.
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MATERIAL AND METHODS |
Drug and chemicals.
Etoposide was obtained from Sigma-Aldrich laboratories (St Quentin
Fallavier, France). Stock solution (20 mmol/L) in dimethyl sulfoxide
(DMSO) was conserved at  20°C for less than 1 month. Further
dilutions were made in culture medium just before use. The final
concentration of DMSO in culture did not exceed 1% (vol/vol), which
was nontoxic to the cells. [2-14C]thymidine (50 mCi/mmol)
was obtained from Amersham (Les Ulis, France). All other chemicals were
purchased from local sources.
Cell lines and culture.
The four human leukemic cell lines, HL60 (promyelocytic), U937
(monocytic), K562 (CML in erythroblastic blast crisis), and KCL22 (CML
in lymphoid blast crisis) were grown in suspension in RPMI 1640 medium
(BioWhittaker, Fontenay-sous-bois, France) supplemented with 10%
(vol/vol) heat-inactivated fetal bovine serum and 2 mmol/L L-glutamine
in an atmosphere of 95% air and 5% CO2. The
human pluripotent UT-7 line, established from a patient with
megakaryocytic leukemia, was cultured in the presence of 10 ng/mL
granulocyte-macrophage colony-stimulating factor (GM-CSF). Transfer of
bcr-abl gene into UT-7 cells was accomplished using a defective
amphotropic p210 retrovirus. The GM-CSF-independent UT7/9 clone was
selected for its high expression of BCR-ABL mRNA as previously
reported.29 Cell viability was determined using the trypan
blue exclusion test. Cells were resuspended at a density of 1.5 × 106/mL in fresh medium before treatment.
Reconstituted cell-free system.
Triton-soluble extracts and nuclear fractions were prepared as
previously described.30 Briefly, cells were washed twice in
10 mL ice-cold phosphate-buffered saline (PBS) without Ca2+
and Mg2+, and once in a buffer containing 150 mmol/L NaCl,
1 mmol/L KH2PO4, 1 mmol/L EGTA, 1 mmol/L
Na3VO4, 5 mmol/L MgCl2, and 10%
glycerol (pH 7.2). Then, the cells were incubated for 10 minutes on ice in the same buffer containing 0.3% Triton X-100 (Sigma,
Aldrich, France) before centrifugation (2,000g for 10 minutes
at 4°C) and the supernatants collected were considered as
triton-soluble extracts. Protein concentration in the supernatants was
determined using the bicinchoninic acid method.31 Pellets
(nuclei) were washed twice in the lysis buffer without Triton X-100.
Triton-soluble extracts (500 µL) from untreated or treated cells were
incubated for 30 minutes at 37°C in the presence of nuclei from 1.0 × 106 untreated cells and DNA fragmentation was measured
as described below. Controls were performed by incubating nuclei from
untreated cells with triton-soluble extracts from untreated cells, in
the absence or in the presence of 100 µmol/L etoposide.
Quantification of DNA fragmentation.
DNA fragmentation was measured using a previously reported filter
elution assay.32 Exponentially growing cells were
prelabeled by adding 0.02 µCi/mL of [2-14C]thymidine in
the culture medium for 2 days. Approximately 1.0 × 106 [14C]-labeled cells or
[14C]-labeled nuclei were loaded onto a protein
absorbing filter (Polyvinylidene fluorure filters, 0.65 µM pore size,
25 mm diameter; Durapore membrane, Millipore, St Quentin, France).
After washing, lysis was performed with 5 mL of LS10 buffer (0.2%
sodium sarkosyl, 2 mol/L NaCl, 0.04 mol/L EDTA, pH 10.0). Filters were
washed with 7 mL of 0.02 mol/L EDTA, pH 10. DNA was depurinated by
adding 0.4 mL of 1N HCl at 65°C for 45 minutes, then released from
the filters by adding 2.5 mL of 0.4 N NaOH for 45 minutes at room temperature. Radioactivity was counted by liquid scintillation spectrometry in each fraction (wash, lysis, EDTA wash, and filter). DNA
fragmentation was measured as the fraction of disintegrations per
minute in the lysis fraction plus EDTA wash relative to the total
intracellular dpm.
Analysis of DNA fragmentation by agarose gel electrophoresis.
Cellular DNA was extracted by a salting-out procedure as described
previously.33 Electrophoresis was performed in a 1.8% agarose gel in Tris-borate-EDTA buffer (pH 8) at 20 V for 15 hours. After electrophoresis, DNA was visualised by ethidium bromide staining.
Western blot analysis.
After treatment, cells were washed twice in PBS, lysed in lysis buffer
(150 mmol/L NaCl, 1 mmol/L KH2PO4, 1 mmol/L
EGTA, 1 mmol/L Na3VO4, and 5 mmol/L
MgCl2; and 10% glycerol containing PmsF 0.1 mmol/L,
Aprotinin 0.15 U/mL, and Pepstatin 1 µg/mL), and then centrifugated
(15 minutes, 15000g). Fifty micrograms of proteins of
supernatants were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by using a 12% polyacrylamide gel and
electroblotted to PVDF membrane (BioRad, Ivry sur Seine, France). After
blocking nonspecific binding sites, the membrane was incubated for 2 hours at room temperature with anti-human CPP32 or Ich-1L monoclonal
antibody (MoAb) (Transduction Laboratories, Lexington, KY), washed, and
further incubated with horseradish peroxidase-conjugated goat
anti-mouse antibody (Jackson Immunoresearch Laboratories, West Grove,
PA) for 30 minutes at room temperature. Immunoblot was revealed by
using enhanced chemiluminescence detection kit (Amersham) by
autoradiography.
For the PARP and CPP32 cleavage analyses, cells were lysed in SDS-PAGE
sample buffer (125 mmol/L Tris-HCl, pH 6.8; 10% -mercaptoethanol; 2% SDS; 20% glycerol; and 0.003% bromophenol blue) and
cell lysates were subjected to SDS-PAGE on a 8% polyacrylamide gel.
The 116-kD native PARP protein and its 85-kD cleavage
product were detected by immunoblotting with anti-human PARP polyclonal
antibody (Vic. 5; kindly given by Dr G. De Murcia, Ecole
supérieure de biotechnologies, Strasbourg, France)
and horseradish-peroxidase conjugated anti-rabbit antibody (Amersham)
as described above. A rabbit polyclonal anti-apopain/CPP32-p17 antibody
(kindly provided by Dr D. Nicholson, Merck Center for Therapeutic
Research, Pointe Claire, Daval, Quebec, Canada) that recognizes both CPP32 proenzyme and its p19 and p17
subunits34 was used to detect CPP32 activation in apoptotic
U937 cells.
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RESULTS |
Etoposide-induced apoptotic DNA fragmentation is delayed in
bcr-abl+ cell lines.
We used a filter elution assay32 to measure apoptotic DNA
fragmentation induced by continuous exposure to etoposide in the four
human leukemic cell lines. In HL-60 and U937 cells, apoptotic DNA
fragmentation appeared rapidly, beginning after 3 hours of exposure to
100 µmol/L etoposide, and increased dramatically because 80% to
100% of DNA was cleaved after 24 hours of drug exposure (Fig
1A). Conversely, in K562 and KCL-22 cells,
apoptotic DNA fragmentation was barely detected 24 hours after the
beginning of cells exposure to 100 µmol/L etoposide (Fig 1A). DNA
fragmentation was confirmed to be internucleosomal by agarose gel
electrophoresis (Fig 1B). We had previously shown that the levels of
DNA double-strand breaks induced by this concentration of etoposide
were similar in the four cell lines.28 This suggested that
the apoptotic pathway was delayed downstream of DNA double-strand
breaks induction in the two bcr-abl+ resistant cell
lines.
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| Fig 1.
Etoposide-induced apoptotic DNA fragmentation and
expression of Bcl-2/Bax proteins in four human leukemic cell lines. (A) DNA fragmentation was measured by a filter elution assay in HL60 ( ),
U937 ( ), K562 ( ), and KCL-22 ( ) cell lines treated for 24 hours with indicated concentrations of etoposide (VP-16). (B) DNA
fragmentation was identified as internucleosomal by agarose gel
electrophoresis. Cells were treated for indicated times with 100 µmol/L etoposide.
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Delayed apoptosis in bcr-abl+ cell lines is not
related to Bcl-2 and Bax expression.
Bcl-2 overexpression was shown to delay drug-induced apoptosis in a
variety of cell systems. Moreover, the Bcl-2 to Bax ratio was shown to
determine the fate of tumor cell treatment with various cytotoxic
drugs.35,36 Therefore, we analyzed the expression of Bcl-2
and Bax proteins in the tested cell lines
(Fig 2). By using Western blot analysis,
Bcl-2 protein was not detected in K562 cells, whereas its level was
rather similar in the three other cell lines. Bax protein level was
similar in the four cell lines (Fig 2). We also analyzed the expression
of the Bcl-2-related protein Mcl-1, whose expression is prominent in
hematopoietic cells. After 3 hours of treatment with etoposide, Bcl-2
and Mcl-1 expressions were slightly decreased in HL-60 and KCL-22
cells, whereas it was unchanged in U937 cells and Bax remained stable in the four cell lines (Fig 2B). These results indicated that the delay
in etoposide-induced apoptosis in bcr-abl+ compared
with bcr-abl cell lines could not be related to
the Bcl-2 to Bax ratio, nor to Mcl-1 expression.
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| Fig 2.
Expression of Bcl-2, Bax, and Mcl-1 in untreated and
etoposide-treated leukemic cell lines. (A) Western blot analysis of
Bcl-2, Bax, and Mcl-1 basal expression in the four studied cell lines. (B) Western blot analysis of Bcl-2, Bax, and Mcl-1 expression in the
four studied cell lines treated for indicated times with 100 µmol/L
etoposide.
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The nuclei of bcr-abl+ cell lines are sensitive
to apoptotic DNA fragmentation.
To determine whether differences between bcr-abl+
and bcr-abl cell lines were related to
differences in the ability of cell nuclei to undergo apoptotic DNA
fragmentation, we used a cell-free system in which triton-soluble
extracts from etoposide-treated cell lines were incubated with
14C-labeled nuclei from untreated cell
lines.30,32 We observed that triton-soluble
extracts from HL-60 and U937 cells treated for 3 hours with 100 µmol/L etoposide induced apoptotic DNA fragmentation in nuclei from
bcr-abl+ and bcr-abl cell
lines (Fig 3A through D). These results
suggested that the delayed induction of apoptotic DNA fragmentation
could not be related to nuclei changes in bcr-abl+
cell lines. Apoptotic DNA fragmentation was lower in K562 nuclei compared with the three other cell lines. Agarose gel electrophoresis confirmed that DNA fragmentation measured in nuclei from the four studied cell lines treated with triton-soluble extracts from
etoposide-treated HL-60 and U937 cells was internucleosomal DNA
degradation (Fig 3E). Conversely, extracts from
bcr-abl+ cells treated with etoposide in the same
conditions did not induce any DNA fragmentation in nuclei from either
bcr-abl+ or bcr-abl cells
(Fig 3). Thus, the apoptotic pathway that leads to apoptotic DNA
fragmentation is blocked at the cytosolic level in
bcr-abl+ cells.
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| Fig 3.
Apoptotic DNA fragmentation in a cell-free system. (A
through D) Nuclei from the four cell lines indicated on axis in which DNA was labeled with [14C]-thymidine were incubated for
30 minutes at 37°C with either etoposide alone (100 µmol/L,  ) or
triton-soluble extracts from untreated ( ) or etoposide-treated (100 µmol/L for 3 hours,  ) cell lines (A = HL60, B = U937,
C = K562, D = KCL22). Apoptotic DNA fragmentation was measured
by filter elution assay. Results shown are the mean ± SD of two
different experiments performed in triplicate. (E) Agarose gel
electrophoresis of nuclear DNA from indicated cell lines before (lane
1, 3, 5, 7, and 9) and after (lane 2, 4, 6, 8, and 10) a 30-minute
incubation in the presence of triton-soluble extracts from
etoposide-treated U937 cells (100 µmol/L for 3 hours).
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Cleavage of procaspase-3 is delayed in bcr-abl+
cell lines.
Using an MoAb that recognizes the 32-kD precursor form of the
CED-3-related caspase-3 and immunoblot analysis, we investigated the
effects of etoposide treatment on procaspase-3 expression in the four
studied cell lines. A rabbit polyclonal anti-caspase-3-p17 antibody
was used to identify the p19 intermediate cleavage form and the p17
active subunit of caspase-3. Expression of the long isoform of
procaspase-2 (ICH-1L), a protease distinct from CED-3-related caspase,
was studied simultaneously (Fig 4).Procaspase-3 basal expression was observed to be lower in HL-60 cells
than in other cell lines. When HL-60 and U937 cells were treated for 3 hours with 50 µmol/L etoposide, procaspase-3 expression decreased,
the p19 and p17 subunits appeared, and the expression of the long isoform of procaspase-2 remained unchanged. Conversely, when
bcr-abl+ K562 and KCL-22 cells were treated with
100 µmol/L etoposide for 3 hours, the procaspase-3 was cleaved only
48 hours after the beginning of drug treatment (Fig 4). These data
suggested that the apoptotic pathway was delayed upstream of
procaspase-3 activation in the bcr-abl+ cell lines.
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| Fig 4.
Activation of procaspase-3 in etoposide-treated leukemic
cells. Western blot analysis of procaspase-2L (the 45-kD long isoform of ICH-1), procaspase-3 (the 32 kD proenzyme CPP32/Yama/Apopain) and
active caspase-3 fragments (p19 and p17) in cell lines treated for
indicated times with 100 µmol/L etoposide.
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bcr-abl Overexpression in UT7 cells confirms that
p210bcr-abl delays procaspase-3 activation and target
protein cleavage.
Although the resistance of K562 cells to drug-induced apoptosis was
previously shown to be related to BCR-ABL expression, we checked some
of the previously related observations in the GM-CSF-dependent UT7
cells and a bcr-abl-transfected clone, UT7/9 (Fig
5). The sensitivity of UT7 cells to
etoposide-induced apoptosis was lower than that of U937 and HL60 cells.
Nevertheless, drug treatment clearly induced apoptotic DNA
fragmentation that was delayed in bcr-abl-transfected UT7/9
cells. The proteolytic cleavage of the nuclear enzyme PARP is a common
apoptosis-associated event that was related to the proteolytic
activation of caspases.37 Cleavage of both procaspase-3 and
PARP was delayed in UT7/9 cells as compared with UT7 cells, whereas the
level of the long isoform of procaspase-2 remained stable (Fig 5).
These results confirmed that p210bcr-abl-mediated
inhibition of the apoptotic pathway occured upstream of procaspase-3
activation. By use of the cell-free system, we showed that
triton-soluble extracts from U937 cells treated for 3 hours with 100 µmol/L etoposide induced apoptotic DNA fragmentation in nuclei from
UT7/9 cells. This observation confirmed that bcr-abl expression
did not inhibit the ability of nuclear DNA to undergo apoptotic
fragmentation (Fig 6).
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| Fig 5.
p210bcr-abl expression delays apoptosis
upstream of procaspase-3 activation and PARP cleavage. (A) Agarose gel
electrophoresis of DNA from UT7 and UT7/9 cells treated for indicated
times with 100 µmol/L etoposide (UT7, left panel; UT7/9: whole panel)
or deprived from GM-CSF for indicated times (UT7, right panel). (B) Western blot analysis of procaspase-2L (the 45-kD long isoform of
ICH-1), procaspase-3 (the 32-kD proenzyme CPP32/Yama/Apopain), active
caspase-3 fragments and PARP in cell lines treated for indicated times
with 100 µmol/L etoposide (UT7, left panel; UT7/9: whole panel) or
deprived from GM-CSF for indicated times (UT7, right panel).
(Co = control untreated cells = time 0.)
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| Fig 6.
Nuclear DNA fragmentation in the
p210bcr-abl-expressing UT7/9 cells. Nuclei from UT7/9 cells
in which DNA was labeled with [14C]-thymidine were
incubated for 30 minutes at 37°C with buffer alone (Buffer) or 100 µmol/L etoposide alone (VP-16) or triton-soluble extracts from either
untreated (U937) or etoposide-treated (100 µmol/L for 3 hours;
U937/VP) U937 cells. Apoptotic DNA fragmentation was then measured by
filter elution assay. Results are the mean ± SD of two
different experiments performed in duplicate.
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Untreated BCR-ABL-expressing cells do not contain an inhibitor of
the apoptotic pathway triggered by etoposide.
To further explore the mechanisms by which BCR-ABL delays
etoposide-induced apoptosis in leukemic cells, we analyzed the
influence of triton-soluble extracts from untreated
bcr-abl+ cells (K562, KCL22, and UT7/9) on the
ability of extracts from etoposide-treated U937 cells to trigger
apoptotic DNA fragmentation in the nuclei from untreated U937 cells
(Fig 7). Extracts from untreated
bcr-abl cell lines (U937, HL60, and UT7) were
used as control. A mixture of an equal volume of extracts from
untreated cells and etoposide-treated U937 cells (100 µmol/L for 3 hours) was incubated for 30 minutes at 37°C with
[14C]-thymidine-labeled nuclei from untreated U937
cells. DNA fragmentation was measured by the filter elution assay. This
experiment indicated that untreated bcr-abl+ cells
do not contain an inhibitor of the apoptotic pathway triggered by
etoposide, further supporting the observation that the
p210bcr-abl protein inhibits the caspase cascade at or
before the cleavage of procaspase-3.
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| Fig 7.
Lack of apoptosis inhibitory effect of triton-soluble
extracts from untreated bcr-abl+ cell lines. Nuclei
from U937 cells in which DNA was labeled with [14C]-thymidine were incubated for 30 minutes at 37°C
with either triton-soluble extracts from untreated (U937) or
etoposide-treated (100 µmol/L for 3 hours; U-VP) U937 cells or a
mixture or an equal volume of triton-soluble extracts from
etoposide-treated (100 µmol/L for 3 hours) U937 cells and
triton-soluble extracts from untreated cell lines (U-VP + name of
the untreated cell line). Results of a representative experiment are
expressed as the mean ± SD of triplicate measurements standardized to
the U-VP sample (100%).
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| |
DISCUSSION |
Decreased ability to undergo apoptosis in response to drug-induced cell
damage is one of the mechanisms by which tumor cells can escape their
cytotoxic activity.27 Cells which overexpressed v-abl or p210bcr-abl receive all the measurable
damage induced by cytotoxic drugs but are unable to couple this damage
to the apoptotic pathway.7,28 Antisense oligonucleotides
that downregulate p210bcr-abl expression render K562 cells
more susceptible to cell death induction by a variety of cytotoxic
drugs.8,10 These oligonucleotide antisenses also render
Fas-transfected K562 cells more sensitive to Fas-induced
apoptosis.12 Thus, v-abl and
p210bcr-abl act downstream of the drug-target interaction
to prevent the coupling of drug-induced DNA damage to the apoptotic
pathways. This decreased sensitivity to apoptosis induction could
contribute to the progression of CML if resistant cells that are
genetically damaged subsequently proliferate. Therefore, it is
essential to understand the molecular mechanisms by which
p210bcr-abl delays the apoptotic pathway triggered by DNA
damaging agents.
The bcl-2 oncogene is a well-characterized inhibitor of
drug-induced cell death. Apoptosis induction by DNA damaging agents is
suppressed by transfection of cells with the oncogene bcl-2 despite similar levels of drug-induced DNA damage in both parent and
transfected cells.27 The clinical syndromes produced by BCR-ABL expression (CML) and Bcl-2 deregulation (follicular lymphoma) both exhibit an initial indolent phase with uninterrupted
differentiation and are both incurable with conventional doses of
chemotherapy.38 Therefore, p210bcr-abl-mediated
inhibition of cell death could have been mediated by overexpression of
Bcl-2. Actually, we did not observe any relationship between Bcl-2 and
Bax protein levels in both untreated (Fig 1) and treated (not shown)
cell lines and their sensitivity to etoposide-induced cell death.
Accordingly, overexpression of v-abl in a clone of HL-60 cells
in which no Bcl-2 protein could be detected remains capable of inducing
an antiapoptotic state.39,40 Bcl-2 is now known to belong
to a growing family of apoptosis-regulatory gene products which may
either be death antagonists or death agonists and we cannot rule out a
role for other Bcl-2 family members as mediators of
p210bcr-abl-induced inhibition of cell death.
We have shown previously that triton-soluble extracts from
etoposide-treated leukemic cell lines such as HL-60 and U937 activated a nuclear endonuclease that triggered apoptotic DNA fragmentation at
neutral pH in the presence of Mg2+.21 In the
present study, experiments performed in this cell-free system show that
the nuclei from untreated cells that express p210bcr-abl
remain sensitive to triton-soluble extracts from
p210bcr-abl-negative cells treated with etoposide. These
results indicate that the nuclease responsible for apoptotic DNA
fragmentation in these cells is not inhibited by
p210bcr-abl.
Growth factors influence the sensitivity of myeloid cell lines to
apoptosis induced by chemotherapeutic drugs.41
The phosphorylation patterns induced by p210bcr-abl and
growth factors show some similarities. Calphostin C, an inhibitor of
protein kinases such as protein kinase C, restores drug sensitivity in
cells with active v-abl, suggesting a role for protein kinases in the suppression of drug-induced apoptosis by v-ABL and
p210bcr-abl.7 However, drug treatment
induces more rapid apoptosis in p210bcr-abl
UT7 cells cultured in the presence of GM-CSF compared with UT7/9 cells
in the absence of GM-CSF, suggesting that p210bcr-abl
imposes a survival advantage over that imparted by GM-CSF and that
cellular components other than p210bcr-abl are probably
involved in its antiapoptotic effect.9,14-16
Recent evidences indicate that activation of procaspases in apoptosis
occurs via a proteolytic cascade.26 For example, caspase-4 can activate procaspase-1 that, in turn, can process
procaspase-3.42 Under normal physiological conditions,
procaspase-1 (pro-ICE) is activated in monocytes and cleaves
pro-interleukin (IL)-1 to produce mature IL-1 cytokine. Other
downstream procaspases such as procaspase-3 might also be activated by
active caspase-1; nevertheless, the cells do not undergo apoptosis.
This suggests that a threshold of tolerance of active caspases exists
in different cells. The present study indicates that
p210bcr-abl-mediated delay in apoptosis induction is
associated with a delayed activation of procaspase-3 in response to DNA
damage and growth factor-deprivation rather than to an increased
threshold of active caspase-3 tolerance. Human PARP is one of the
specific targets of apoptosis-associated proteolysis.36
Although most caspases cleave this protein in
vitro,22-24,43 kinetic properties of CED-3-related caspases suggest these proteases to be the most involved in PARP cleavage.21,23 Inhibition of PARP cleavage in
p210bcr-abl+ UT7/9 cells treated with etoposide
suggests that p210bcr-abl prevents the activation of all
CED-3-related caspases. Interestingly, the long isoform of caspase-2
is not cleaved and activated in any tested cell line when exposed to
VP-16, indicating that all the caspases are probably not implicated in
the VP-16-induced cell death pathway. Experiments performed in the
cell-free system showed that neither p210bcr-abl nor other
cellular components involved in its antiapoptotic effect could prevent
apoptotic DNA fragmentation triggered by extracts from VP-16-treated
U937 cells, further supporting the conclusion that
p210bcr-abl acts at or upstream the procaspase-3 activation
level.
Inhibition of the etoposide-mediated apoptotic pathway upstream of the
activation of this CED-3-related caspases is in accordance with the
recent observation that p210bcr-abl-mediated resistance to
apoptosis is overcome by cytotoxic T cells44 and NK and LAK
cells.29 These lymphocytes use two systems to induce cell
death in target cells, namely, the granzyme and the Fas systems. The
substantial role played by granzyme B in the former has been confirmed
by targeted gene disruption. Granzyme B is a serine protease with an
unusual specificity for cleaving synthetic substrates after Asp
residues. Granzyme B activates several CED-3-related procaspases
including procaspase-3 and procaspase-7, whereas procaspase-1 is not a
substrate for granzyme B.45 Several recent studies
established that Bcl-2 and Bcl-XL function upstream of the
CED-3-related caspases. The present study shows that
p210bcr-abl similarly delays drug-induced apoptosis
upstream of these proteases.
Several methods were proposed to overcome drug resistance of CML cells.
Tyrosine kinase inhibitors inhibit the growth of the murine
IL-3-dependent myeloid 32Dc13(G) cell line as well as a subclone
transformed to IL-3-independent growth by retroviral transduction and
expression of BCR-ABL. However, these compounds induced apoptosis in
the parental cells and necrosis in bcr-abl-transformed cells,
confirming that BCR-ABL could suppress apoptotic signal transduction.11 Combination of antisense oligonucleotides
that downregulate BCR-ABL protein with a conventional chemotherapeutic agent such as mafosfamide was shown to be an efficient strategy to
eliminate CML cells in mice.46 The present study indicates that targeting CED-3-related caspases could be another efficient way
to overcome drug resistance of CML cells by acting downstream of the
apoptotic pathway inhibition.
| |
FOOTNOTES |
Submitted July 23, 1997;
accepted November 10, 1997.
Supported by grants from the Burgundy, Saône et Loire and Yonne
Comittees of the Ligue Nationale Contre le Cancer, the Association pour
la Recherche contre le Cancer (ARC#4075) and the Conseil Régional
de Bourgogne.
Address reprint requests to Eric Solary, MD, INSERM CJF 94-08, Faculté de Médecine, 7, Blvd Jeanne d'Arc, 21033 DIJON
Cedex, France.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract