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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 10 (November 15), 1999:
pp. 3523-3530
Defective Apoptotic Signal Transduction Pathway Downstream of Caspase-3
in Human B-Lymphoma Cells: A Novel Mechanism of Nuclear Apoptosis
Resistance
By
Yoshinari Kawabata,
Makoto Hirokawa,
Atsushi Kitabayashi,
Takahiro Horiuchi,
Jun Kuroki, and
Akira B. Miura
From the Department of Internal Medicine III, Akita University School
of Medicine, Akita, Japan.
 |
ABSTRACT |
Mitochondria play a central role in controlling apoptosis, and
activation of the caspase cascade appears to be crucial event during
the apoptotic process. Human B lymphoma Raji cells are resistant to
nuclear apoptosis induced by various stimuli. Using this cell line, we
have asked whether reduction of the mitochondrial transmembrane
potential and activation of caspase-3 are sufficient to induce DNA
fragmentation during the apoptotic process. After stimulation with
cell-permeable C2-ceramide or mitochondrial permeability transition
(PT) inducers, not only apoptosis-sensitive cell lines (HL-60, Jurkat,
and Daudi cells), but also Raji cells showed reduction of the
mitochondrial transmembrane potential (  m), activation of
caspase-3, and loss of clonogenic potential. However, Raji cells did
not show detectable levels of nuclear apoptosis (DNA degradation). In a
cell-free system, cell lysates from tetra-butylhydroperoxide (t-BHP)-treated HL-60 cells induced DNA degradation of Raji nuclei, whereas cell lysates from t-BHP-treated Raji cells failed to induce DNA degradation in either apoptosis-sensitive cell lines or
apoptosis-resistant Raji cells. Cleavage of DFF-45, which is a
downstream target molecule for caspase-3, was observed in Raji cells as
well as in apoptosis-sensitive Daudi cells. These results indicate that
there is a defective apoptotic pathway in the cytoplasm downstream of
caspase-3 in Raji cells.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
APOPTOSIS IS A CRUCIAL pathway not only
for physiological mammalian development, but also for tumor cell
killing by cytotoxic T cells, natural killer cells,1
irradiation or cytotoxic drugs.2-5 Although
chemoradiotherapy or immunological therapy has been successful in the
treatment of several human cancers, such as hematopoietic malignancies,
and rarely in melanoma, these approaches have been mostly unsuccessful
in most other malignant tumors. Resistance to apoptosis has been
established as one important mechanism of treatment failure, in
addition to altered drug metabolism in cancer cells conferred through
the P-glycoprotein encoded by MDR1, redox detoxifying action of
glutathione, enhanced hepatic P450 activity, and mutation of
topoisomerase II.6 Comparing the apoptotic process in
apoptosis-sensitive with that in apoptosis-resistant cancer cells may
provide new insights into treatment resistance in malignant tumors.
The apoptotic process can be divided into the cell-type-specific
induction phase, which is death-stimulus-dependent, and the common
effector/degradation phase.7 Mitochondria play a central role in determining whether to die during the common apoptotic signaling pathway, and the decision to die is transduced via the caspase cascade, subsequently leading to DNA degradation. Disruption of
the mitochondrial transmembrane potential ( m) and subsequent DNA
fragmentation occur in various cell types after distinct types of
apoptosis-inducing stimulation.8-10 In addition, disruption of the mitochondrial transmembrane potential has been shown to lead to
an irreversible apoptotic process.8
We have previously reported that cell-permeable ceramide inhibits the
growth of B-lymphoma Raji cells by inducing G0/G1 cell cycle arrest but
not apoptosis.11 Since completing this report, we have also
found that antitumor agents such as doxorubicin and etoposide could not
induce DNA fragmentation in Raji cells.
We have been addressing the question of what is required for the
apoptotic process by asking what is missing in the apoptotic pathways
of this cell line. In this report, we demonstrate that reduction of
mitochondrial transmembrane potential and activation of caspase-3 are
not sufficient to induce DNA fragmentation in Raji cells and show
evidence that certain molecules downstream of caspase-3 appear to be
lacking or not functioning properly in this particular cell line. We
also discuss the role of DNA fragmentation factor (DFF) during the
apoptotic process in this cell line.
| |
MATERIALS AND METHODS |
Cell lines.
HL-60 (acute myelogenous leukemia), U937 (monoblastic leukemia), Jurkat
(T-cell leukemia), NALM-6 (B-cell leukemia), K562 (chronic myelogenous
leukemia), Ramos (Epstein-Barr virus [EBV]-negative Burkitt's lymphoma), Daudi (EBV-positive Burkitt's lymphoma), and
Raji (EBV-positive Burkitt's lymphoma) cells were provided by Fujisaki
Cell Center (Okayama, Japan) and were maintained in RPMI 1640 medium
supplemented with mycoplasma-free heat-inactivated 10% fetal calf
serum (FCS; Hyclone, Logan, UT), 2 mmol/L L-glutamine, 100 U/mL
penicillin and 100 µg/mL streptomycin at 37°C. Exponentially growing cells were used in this study, and viability was determined by
trypan blue dye exclusion test.
Reagents.
Mitochondrial transmembrane permeability transition (PT) inducers,
tetra-butylhydroperoxide (t-BHP) and diamide, were purchased from Sigma
(St Louis, MO). A cell-permeable ceramide analog, C2-ceramide, was
purchased from Matreya (Pleasant Gap, PA). Rhodamine 123 (Rh123) was
purchased from Sigma. Purified anti-Fas (CD95) monoclonal antibody
(MoAb; IgM, clone CH-11) and fluorescein isothiocyanate (FITC)-conjugated anti-Fas MoAb (IgG1, clone DX2) were obtained from
MBL (Nagoya, Japan) and Becton Dickinson (San Jose, CA), respectively.
Measurement of the mitochondrial transmembrane potential.
A cationic lipophilic fluorochrome, Rh123, was used to measure the
mitochondrial transmembrane potential (m), as previously reported.12 Briefly, cells were treated with mitochondrial
transmembrane PT inducers or C2-ceramide and were then incubated with
Rh123 at a final concentration of 10 µmol/L for 30 minutes. After
washing twice with phosphate-buffered saline (PBS), fluorescence
intensity was determined by a flow cytometer. Fluorescence intensity
was also monitored by fluorescence microscopy.
DNA fragmentation.
To determine the extent of DNA fragmentation, agarose gel
electrophoresis and cell-cycle analysis using a flow cytometer were used in this study. The details of the procedures have been described elsewhere.11 In some experiments, TdT-mediated dUTP nick
end labeling (TUNEL) assay was performed using an In situ Apoptosis Detection Kit (TaKaRa, Osaka, Japan). For DNA electrophoresis, 2 × 106 cells were washed twice with PBS and were lysed
in 200 µL of lysis buffer (50 mmol/L Tris-HCl [pH 8.0]/10 mmol/L
EDTA/0.5% lauroylsarcosinate). Cell lysates were incubated with
proteinase K (0.5 mg/mL) for 1 hour at 50°C. RNase A (0.25 mg/mL;
Nippongene, Osaka, Japan) was then added and incubated for an
additional 1 hour at 50°C. Extracted DNA was loaded in 2% agarose
gels and electrophoresed in 1× TBE buffer. DNA was visualized by
soaking the gels in TBE buffer containing 1 µg/mL ethidium bromide
for 30 minutes.
Flow cytometric analysis of DNA loss was performed as follows. After
treatment with the various stimuli, cells were washed twice with PBS
and were then fixed with 70% ethanol/PBS on ice. After fixation, the
medium was removed by centrifugation, 500 µL of PBS was added to each
sample, and the cells were treated by DNase-free RNase A (20 µg/mL)
for 45 minutes at 37°C. The cells were then stained with 500 µL
of propidium iodide (PI; 50 µg/mL) for 30 minutes at room temperature
and subjected to flow cytometry. Flow cytometric analysis was performed
using a Cytron (Ortho Diagnostics, Tokyo, Japan) with a
cell cycle program (Version 1.4). DNA degradation in each cell was
defined as a nucleus containing less than 2N DNA.
In some experiments, cell viability was determined by vital staining
with PI.
Isolation of intact nuclei.
Intact nuclei were prepared according to the method previously
reported.13 Untreated HL-60 and Raji cells (5 × 106) were washed twice with cold PBS and pelleted by
centrifugation at 800g for 5 minutes. The cells were incubated
in STKM buffer (0.25 mol/L sucrose/50 mmol/L Tris-HCl [pH 7.5]/25
mmol/L KCl/5 mmol/L MgCl2/0.25% Triton X-100) for 30 minutes on ice. After incubation, nuclei were pelleted by
centrifugation at 800g for 10 minutes. Sedimental nuclei were
washed once in STKM buffer. The release of cytosol-free nuclei was
monitored by phase contrast microscopy.
Preparation of cell lysates.
HL-60 and Raji cells (1 × 107) were exposed to 500 µmol/L t-BHP for 4 hours at a density of 1 × 106/mL. The cells were then washed once with cold PBS and
were incubated in STKM buffer for 30 minutes on ice. After incubation,
nuclei were pelleted by centrifugation at 800g for 10 minutes.
Supernatants were then centrifuged at 2,600g for 15 minutes and
were used as cell lysates in a cell-free system. Nuclei-free cytosol
was confirmed by phase contrast microscopy.
Analysis of DNA degradation in a cell-free system.
Intact nuclei from untreated HL-60 or Raji cells (5 × 105) were suspended in 150 µL of each cell lysate derived
from untreated or t-BHP-treated cells (1 × 106 cell
equivalent). After adding EGTA at a final concentration of 5 mmol/L,
the reaction mixtures were incubated at 37°C for the indicated
periods. After incubation, the reaction mixtures were centrifuged at
800g for 5 minutes. Pellets were washed once with STKM buffer
and were fixed with cold 70% ethanol/PBS. The medium was removed by
centrifugation, and 500 µL of STKM buffer was added to each sample.
The cells were treated by DNase-free RNase A (20 µg/mL) for 45 minutes. The nuclei were then stained with 500 µL of PI (50 µg/mL)
for 30 minutes at room temperature and subjected to flow cytometry.
Immunoblotting for caspase-3 and DFF-45.
After washing twice with PBS, 1 × 106 cells were
resuspended in 20 µL of RIPA buffer (1% NP40/0.1% sodium
deoxycholate/150 mmol/L NaCl/50 mmol/L Tris-HCl [pH 7.5]/1 mmol/L
phenylmethylsulfonyl fluoride [PMSF]/20 U/mL aprotinin)
on ice. After centrifugation at 10,000g at 4°C for 10 minutes, supernatants were harvested and were subjected to 12.5%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
under reducing conditions. Transfer of the resolved proteins and
visualization of the proteins were previously described in
detail.14 Mouse antihuman caspase-3 and mouse antihuman
DFF-45 MoAbs were purchased from MBL. Polyclonal rabbit anti-caspase-3
antibody reacting with both the 32-kD unprocessed pro-caspase-3 and
the 17-kD subunit of the active caspase-3 was obtained from PharMingen
(San Diego, CA).
Assay for caspase-3 activity.
Activity of intracellular caspase-3 was determined using a
PhiPhiLux-G6D2 Kit15,16 (OncoImmunin, Inc, College Park,
MD) according to the manufacturer's instructions. Cells
(5 × 105) were exposed to 500 µmol/L t-BHP for the
indicated intervals at 37°C at a density of 1 × 105/mL. After incubation, 50 µL of 10 µmol/L
fluorogenic caspase-3 substrate solution and 5 µL of FCS were added
to each cell pellet and centrifuged at 800g for 5 minutes. The
cells were then incubated at 37°C for 60 minutes. After incubation,
500 µL of ice-cold flow cytometry dilution buffer was added to this
reaction mixture, and the cells were subjected to flow cytometry.
Reverse transcriptase-polymerase chain reaction
(RT-PCR) for DFF-45.
Total cellular RNA was prepared according to the method previously
reported.17 cDNA synthesis was performed using a
First-Strand Synthesis Kit (Pharmacia, Uppsala, Sweden).
The primers 5'-TGTGGCATTGGCTAGTAATGAGA-3' and
5'-ACTAGATAAGCTCAGCTCTGGAG-3' were used to amplify the
DFF-45 cDNA. The PCR products include the 2 cleavage sites of
caspase-3, DETD (aa117) and DAVD (aa224). Thirty PCR cycles were
performed at 94°C for denaturation, 56°C for annealing, and
72°C for extension reaction. PCR products were loaded onto 2%
agarose gels and electrophoresed in 1× TBE buffer. Gels were
stained with ethidium bromide and visualized under UV light.
Sequencing of DFF-45 cDNA.
The PCR products of DFF-45 cDNA were directly sequenced using a Dye
Terminator Cycle Sequencing Kit (PE Applied Biosystems, Foster City,
CA). PCR products were extensively purified using Centricon-100 concentrator columns, and the same forward and reverse primers were used for sequencing chemistry. Sequence analysis was
performed using an Applied Biosystems 377A automatic DNA sequencer (Applied Biosystems, Foster City, CA).
Assay for clonogenic cell growth of tumor lines.
Cells (1,000 cells/mL) were cultured in a 35-mm petri dish in semisolid
culture medium consisting of 0.8% methylcellulose/RPMI 1640/20% FCS
at 37°C. After 7 days of culture, a cell cluster containing more
than 50 cells was counted as a colony under an inverted microscope.
| |
RESULTS |
Cell-permeable ceramide and DNA-damaging agents failed to induce
nuclear apoptosis (DNA fragmentation) in Raji cells.
We have previously reported that a cell-permeable ceramide analogue can
inhibit the growth of Raji cells but not induce nuclear apoptosis.11 We have extended this observation to learn
whether other apoptosis-inducing stimuli cause DNA fragmentation in
this particular cell line. As shown in
Table 1, not only C2-ceramide, but also
antitumor agents, such as doxorubicin and etoposide, failed to induce
DNA degradation in Raji cells, whereas in other cell lines these
reagents induced apoptosis. Moreover, Raji cells did not undergo
apoptosis in response to anti-Fas MoAb treatment, which could induce
apoptosis in Jurkat cells. To define the mechanisms of resistance to
apoptosis in Raji cells, we first focused on the alteration of the
mitochondrial transmembrane potential and the activation of caspase-3.
Loss of the mitochondrial transmembrane potential did not result in
DNA fragmentation in Raji cells.
We first examined whether a cell-permeable ceramide analogue might
induce reduction of the mitochondrial transmembrane potential in
apoptosis-resistant Raji cells. After treatment with C2-ceramide, disruption of the mitochondrial transmembrane potential and subsequent apoptosis were observed in HL-60, U937, Jurkat, and Daudi cells. Interestingly, C2-ceramide reduced the mitochondrial transmembrane potential in Raji cells, although it did not induce DNA degradation (Fig 1). The MTT assay showed that Raji
cells were more resistant to ceramide treatment than were other cell
lines (Table 2), but approximately 20% of
the C2-ceramide-treated Raji cells were found to be dead after 48 hours
of culture by trypan blue staining (data not shown). As shown in
Table 3, Raji cells lost their clonogenic potential after treatment with cell-permeable ceramide, suggesting that
m disruption is the point of no return for cell death, irrespective of death type.
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| Fig 1.
Reduction of the mitochondrial transmembrane potential by
a cell-permeable ceramide in Raji cells. Cells were cultured with
either C2-ceramide (20 µmol/L) or C2-dehydroceramide (20 µmol/L)
for the indicated periods. After incubation, cells were harvested for
measurement of the mitochondrial transmembrane potential.
|
|
Loss of the mitochondrial transmembrane potential has been shown to
result in mitochondrial permeability transition, which then causes
mitochondrial release of apoptogenic proteins.17 Thus, we
asked whether forced mitochondrial permeability transition would cause
DNA fragmentation in Raji cells. We used Daudi cells as a control in
this experiment, because the EBV genome was integrated in both Raji and
Daudi cells. After the treatment with diamide, Raji cells showed a
decrease of incorporation of a cationic lipophilic fluorochrome, Rh123,
to the same degree as apoptosis-inducible Daudi cells
(Fig 2). Other apoptosis-sensitive cells,
such as HL-60, U937, and Jurkat cells, showed similar results (data not shown). Apoptosis, which was judged by the presence of cells with hypodiploid DNA content on flow cytometry, was observed in Daudi cells
as early as 6 hours after treatment with diamide, and almost all cells
showed DNA degradation at 24 hours of incubation with diamide
(Fig 3). Other apoptosis-sensitive cells,
such as HL-60, U937, Jurkat, K562, NALM-6, and Ramos cells, also showed
similar results (Table 1). In contrast, the ratio of apoptotic cells was not significant, even 48 hours after stimulation with diamide in
Raji cells (Fig 3). This observation was confirmed by using another PT
inducer, t-BHP, and DNA electrophoresis analysis. t-BHP failed to
induce DNA fragmentation in Raji cells, whereas t-BHP induced apoptosis
in HL-60 and Jurkat cells (Fig 4).
Moreover, we have performed TUNEL assay to confirm that Raji cells are
resistant to nuclear apoptosis (Fig 5).
However, vital staining of Raji cells with PI showed that Raji cells
died after t-BHP. These results indicate that mitochondrial dysfunction
induced by PT inducers does not lead to DNA degradation, but rather
leads to cell death in Raji cells.
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| Fig 2.
Disruption of the mitochondrial transmembrane potential
by PT inducers. Raji and Daudi cells were cultured in the presence or
absence of 1 mmol/L diamide for the indicated periods. Reduction of the
mitochondrial transmembrane potential was observed in both Daudi and
Raji cells. Similar results were obtained with t-BHP (data not
shown).
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| Fig 3.
Failure to induce DNA degradation in Raji cells by PT
inducers. After treatment with 1 mmol/L diamide, the cells were fixed,
stained with PI, and subjected to flow cytometry. This experiment was
repeated 3 times with similar results. The use of t-BHP also gave
similar results (data not shown).
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| Fig 4.
Induction of DNA fragmentation in apoptosis-sensitive
HL-60 and Jurkat cells but not in apoptosis-resistant Raji cells. The
cells were treated with 50 µmol/L t-BHP for 4 hours at 37°C and
were then harvested for DNA isolation. Characteristic apoptotic DNA
ladders were observed in HL-60 and Jurkat cells but not in Raji cells.
This experiment was repeated more than 3 times with similar results.
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| Fig 5.
TUNEL assay and cell-cycle analysis to determine the
presence of DNA fragmentation. Daudi and Raji cells were stimulated
with 50 µmol/L t-BHP for 6 hours at 37°C. Apoptosis-permissive
Daudi cells, but not apoptosis-resistant Raji cells, were nick-end
labeled by TdT. Viability was determined by staining the unfixed cells
with PI.
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t-BHP can induce activation of caspase-3 in nuclear
apoptosis-resistant Raji cells.
Recently, it has been suggested that reduction of the mitochondrial
transmembrane potential and activation of the caspase cascade are
common events leading to DNA degradation during the apoptotic
process.7,18-23 Apoptogenic proteins released from mitochondria have been shown to activate the caspase
cascade.24,25 We asked whether t-BHP could induce the
activation of caspase after the induction of mitochondrial
transmembrane potential loss in Raji cells. Caspase-3 is a protease
that is activated downstream of caspase-1 during
apoptosis25 and cleaves substrates such as poly
(ADP-ribose) polymerase, actin, fodrin, and lamin.26,27 Therefore, we determined the expression of caspase-3 protease in Raji
cells. Immunoblotting analysis showed that Raji cells expressed a level
of procaspase-3 protein similar to that of other apoptosis-sensitive
cells (data not shown).
We then asked whether PT inducers induce the activation of caspase-3 in
Raji cells. To examine the activity of caspase-3, we performed flow
cytometric analysis using a fluorogenic substrate for caspase-3 that
released fluorescence in proportion to caspase-3 activity in living
cells. As shown in Fig 6, activation of
caspase-3 was observed in t-BHP-treated Raji cells. To confirm this
finding, we also performed immunoblotting to detect cleavage of
procaspase-3 in Raji cells. After treatment with t-BHP, a decrease of
p32 procaspase-3 protein expression was observed and simultaneously p17
active caspase-3 was detected in Raji cells as well as Daudi cells
(Fig 7).
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| Fig 6.
Activation of caspase-3 in Raji cells by t-BHP. HL-60 and
Raji cells were cultured in the presence or absence of 500 µmol/L
t-BHP for the indicated periods. Intracellular caspase-3 activity was
assessed by flow cytometric analysis using a PhiPhiLux-G6D2 kit.
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| Fig 7.
Cleavage of procaspase-3 in Raji cells. Daudi and Raji
cells were stimulated with 500 µmol/L t-BHP for the indicated
periods, and then cell lysates were prepared. Samples were loaded onto
13% SDS-PAGE gels. Transferred proteins on polyvinylidene difluoride
(PVDF) membranes were reacted with polyclonal rabbit
anti-caspase-3 antibody (PharMingen). This experiment was repeated 3 times with similar results.
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Induction of DNA degradation of isolated Raji nuclei with a
cell-free cytosolic extract from t-BHP-treated HL-60 cells.
As described above, the activation of caspase-3 did not lead to DNA
degradation in Raji cells. We raised the question of whether there
might be some defects in Raji nuclei that prevent induction of DNA
degradation. We employed a cell-free system using isolated nuclei and
cytosolic extracts. The cell-free cytosolic extract from t-BHP-treated
HL-60 cells induced DNA degradation not only in HL-60 nuclei, but also
in Raji nuclei (Fig 8A). In contrast, the
cytosolic extract from t-BHP-treated Raji cells did not induce DNA
degradation in either Raji or HL-60 nuclei (Fig 8B). These results
indicate that sufficient apoptotic signals generated in the cytoplasm
of apoptosis-permissive cells induce DNA degradation in Raji nuclei and
that apoptotic signals in the cytoplasm of Raji cells treated with
t-BHP are not sufficient to induce DNA degradation in nuclei of
apoptosis-permissive cells. These data suggest that cytoplasmic
downstream target molecules for caspase-3, which should be activated
during the apoptotic process, might be missing or functionally
defective in Raji cells.
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| Fig 8.
Induction of DNA degradation in Raji nuclei by the
t-BHP-treated HL-60 cytosolic extract. (A) The t-BHP-treated HL-60
cytosolic extract induced DNA degradation in Raji nuclei as well as in
HL-60 nuclei. (B) The cell-free cytosolic extract from t-BHP-treated
Raji cells did not induce DNA degradation in either HL-60 or Raji
nuclei.
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Expression and cleavage of DFF-45 in Raji cells.
The DFF, which induces DNA fragmentation after it is activated by
caspase-3, has been identified in human HeLa and U937
cytoplasm.28 The DFF has been reported to be a
heterodimeric protein of 40- and 45-kD subunits, of which the 45-kD
subunit (DFF-45) can be cleaved by caspase-3 into smaller polypeptides
of 30 and 11 kD.28 The 40-kD subunit (DFF-40) remains
intact after stimulation by active caspase-3. Caspase-3 cleaves DFF-45
at the 2 cleavage sites to generate an active factor that produces DNA
fragmentation without further requirement for caspase-3 or other
cytosolic proteins.28 The amino acid sequence of DFF-45 was
identified as a novel protein, of which an open reading frame of 331 amino acids was predicted from its cDNA sequence.28
Furthermore, Liu et al28 reported that the
sequences of the 2 cleavage sites of DFF-45 for active caspase-3 were
DETD (aa177) and DATD (aa224), respectively, in HeLa and U937 cells. We
tested the possibility that DFF-45 might be deficient or mutated in
Raji cells. To determine the expression of DFF-45 in Raji cells, we
first performed RT-PCR using primers that were designed to
amplify the sequence spanning the 2 caspase-3 cleavage sites. Agarose
gel electrophoresis of these PCR-amplified cDNAs showed that expression
of DFF-45 mRNA was detected in Raji cells with the same migration
pattern as in other cells (data not shown). The cDNA sequence of DFF-45
in Raji cells was identical to that of U937 cells, and it included the
same sequences of the 2 known cleavage sites for caspase-3 reported in
HeLa and U937 cells (data not shown). In fact, the cleavage of DFF-45
was confirmed by immunoblotting analysis in Raji cells as well as
apoptosis-sensitive Daudi cells (Fig 9).
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| Fig 9.
Cleavage of the DFF-45 protein after mitochondrial
dysfunction. After treatment with t-BHP (500 µmol/L), smaller protein
bands, which were reactive with anti-DFF45 MoAb, were detected in both
Daudi and Raji cells.
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| |
DISCUSSION |
Mitochondria play a crucial role in apoptosis.7 During the
apoptotic process, mitochondria release several apoptogenic molecules
to activate the downstream apoptotic signal transduction pathways,
including apoptosis-inducing factor (AIF)23 and cytochrome C.29 AIF is released from mitochondria after the reduction
of the mitochondrial transmembrane potential and subsequent membrane permeability transition.12,18 AIF then activates
caspases.24 Cytochrome C is released during the early phase
of apoptosis before the occurrence of mitochondrial membrane potential
loss,30,31 which appears to be regulated independently from
mitochondrial dysfunction. Cytochrome C also activates
caspases.29 Caspase-3 is a protease that cleaves various
cellular proteins, such as poly (ADP-ribose) polymerase, actin, fodrin,
and lamin during apoptosis,26,27 and has been postulated to
be an "executioner" protein activated by the caspase
cascade.31
In this report, we have demonstrated that reduction of mitochondrial
transmembrane potential and activation of caspase-3 do not lead to DNA
fragmentation in human B-lymphoma Raji cells. The results from
experiments using a cell-free system suggest that there is a defective
apoptotic pathway in the cytoplasm downstream of caspase-3 in this
particular cell line. Disruption of the mitochondrial transmembrane
potential has been detected in various cell types by distinct
apoptosis-inducing stimuli, and subsequently occurring nuclear
apoptosis cannot be dissociated from this biochemical event in each
system.7,9,18,32,33 To our knowledge, this is the first
report describing the model in which loss of the mitochondrial
transmembrane potential and activation of caspase-3 do not lead to DNA
degradation in living cells. However, Raji cells lose their
proliferative capacity after ceramide treatment, suggesting that
m disruption is an irreversible step of cell death programs.
There is an interesting report that Raji and Ramos cells show distinct
types of cell death in response to polyunsaturated fatty
acids.34 Raji and Ramos cells die by necrosis
and apoptosis, respectively. We are currently investigating the cell
death mechanisms in Raji cells in response to ceramide or mitochondrial
permeability transition inducers.
The signal transduction pathway between the caspase cascade and the DNA
degradation phase in humans has not been fully elucidated. Human DFF
has recently been identified as a protein that is activated by
caspase-3 and induces nuclear DNA fragmentation without any requirement
for other cytosolic proteins.28 DFF is a heterodimeric protein consisting of 45- and 40-kD subunits (designated DFF-45 and
DFF-40). DFF-45 has been cloned, and it is cleaved by caspase-3 at the
sequences DETD (aa 117) and DAVD (aa 224).28 We performed partial sequencing of DFF-45 cDNA in Raji and U937 cells and could not
find any difference in the sequence, which is identical to that
previously reported in HeLa and U937 cells.28 We also
confirmed the cleavage of DFF-45 in Raji cells. These results suggest
that certain apoptotic pathways after the cleavage of DFF-45 are
defective in this cell line.
Murine caspase-activated deoxyribonuclease (CAD) and its inhibitor
(ICAD) have recently been identified and cloned. 35The
amino acid sequence of human DFF-45 is highly homologous to that of
murine ICAD,35 suggesting that human DFF-45 could be a
counterpart of murine ICAD. Murine CAD exists as a complex with ICAD in
the cytoplasm and caspase-3 cleaved ICAD at the 2 cleavage sites to
allow CAD to enter the nuclei and induce DNA
fragmentation.35,36 Human DFF-40 has been recently
cloned,37 and the homology between DFF-40 and murine CAD
has been reported to be 71%.37 It is possible that there
might be structural and/or functional defects in DFF-40 in Raji cells,
and this issue should be addressed in the near future.
p53 and Bcl-2 are extensively investigated molecules that modulate
apoptotic cell death, and their functional alteration is thought to be
involved in resistance to apoptosis in human cancers.38-40 In our previous report, it was demonstrated that the expression of p53
protein was normal, and the expression of Bcl-2 was even less in Raji
cells compared with HL-60 or U937 cells, which are known to be
sensitive to apoptosis.11 Because the apoptotic machinery
in the nuclei of Raji cells was normal, alterations in p53 do not
appear to be the cause of apoptosis resistance. Although the structural
and/or functional defects downstream of caspase-3 in Raji cells remain
to be elucidated, exploration of the mechanisms will lead to a new
model of resistance to nuclear apoptosis.
| |
ACKNOWLEDGMENT |
The authors thank Fujisaki Cell Center for providing us with cell lines.
| |
FOOTNOTES |
Submitted October 22, 1998; accepted July 10, 1999.
Supported in part by grants from the Japan Research Foundation for
Clinical Pharmacology and the Ministry of Welfare, Science and Culture,
Japan to M.H.
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 Makoto Hirokawa, MD, PhD, Department of
Internal Medicine III, Akita University School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan; e-mail: hirokawa{at}med.akita-u.ac.jp.
| |
REFERENCES |
1.
Nagata S:
Apoptosis death factor.
Cell
88:355, 1997[Medline]
[Order article via Infotrieve]
2.
Kaufmann SH:
Induction of endonucleolytic DNA cleavage in human acute myelogenous leukemia cells by etoposide, camptothecin, and other cytotoxic anticancer drugs.
Cancer Res
49:5870, 1989[Abstract/Free Full Text]
3.
Gunji H, Kharbanda S, Kufe D:
Induction of internucleosomal DNA fragmentation in human myeloid leukemia cells by 1- -D-arabinofuranosylcytosine.
Cancer Res
51:741, 1991[Abstract/Free Full Text]
4.
Evans D, Dive C:
Effects of cisplatin on the induction of apoptosis in proliferating hepatoma cells and nonproliferating immature thymocytes.
Cancer Res
53:2133, 1993[Abstract/Free Full Text]
5.
Borner MM, Myers CE, Sartor O, Sei Y, Toko T, Trepel JB, Schneider E:
Drug-induced apoptosis is not necessary dependent on macromolecular synthesis or proliferation in the p53-negative human prostate cancer cell line.
Cancer Res
55:2122, 1995[Abstract/Free Full Text]
6.
Fisher DE:
Apoptosis in cancer therapy: Crossing the threshold.
Cell
78:539, 1994[Medline]
[Order article via Infotrieve]
7.
Kroemer G, Zamzami N, Susin AS:
Mitochondrial control of apoptosis.
Immunol Today
18:44, 1997[Medline]
[Order article via Infotrieve]
8.
Zamzami N, Marchetti P, Castedo M, Zanin C, Vayssiere J, Petit XP, Kroemer G:
Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo.
J Exp Med
181:1661, 1995[Abstract/Free Full Text]
9.
Casteds M, Hirsch T, Susin A:
S, Zamzami N, Marchetti P, Macho A, Kroemer G: Sequential acquisition of mitochondrial and plasma membrane alterations during early lymphocyte apoptosis.
J Immunol
157:512, 1996[Abstract]
10.
Marchetti P, Castedo M, Susin A:
S, Zamzami N, Hirsch T, Macho A, Haeffner A, Hirsch F, Geuskens M, Kroemer G: Mitochondrial permeability transition is a central coordinating event of apoptosis.
J Exp Med
184:1155, 1996[Abstract/Free Full Text]
11.
Kuroki J, Hirokawa M, Kitabayashi A, Lee M, Horiuchi T, Kawabata Y, Miura AB:
Cell-permeable ceramide inhibits the growth of B lymphoma Raji cells lacking TNF- -receptors by inducing G0/G1 arrest but not apoptosis: A new model for dissecting cell-cycle arrest and apoptosis.
Leukemia
10:1950, 1996[Medline]
[Order article via Infotrieve]
12.
Shimizu S, Eguchi Y, Kamiike W, Waguri S, Uchiyama Y, Matsuda H, Tsujimoto Y:
Bcl-2 blocks loss of mitochondrial membrane potential while ICE inhibitors act different step during inhibition of death induced by respiratory chain inhibitors.
Oncogene
13:21, 1996[Medline]
[Order article via Infotrieve]
13.
Yoshida A, Takauji R, Inuzuka M, Ueda T, Nakamura T:
Role of serine and ICE-like proteases in induction of apoptosis by etoposide in human leukemia HL-60 cells.
Leukemia
10:821, 1996[Medline]
[Order article via Infotrieve]
14.
Hirokawa M, Kitabayashi A, Kuroki J, Miura AB:
Signal transduction by B7/BB1 expressed on activated T lymphocytes: Cross-linking of B7/BB1 induces protein tyrosine phosphorylation and synergizes with signaling through T-cell receptor/CD3.
Immunology
86:155, 1995[Medline]
[Order article via Infotrieve]
15.
Packard BZ, Toptygin DD, Komoriya A, Brand L:
Profluorescent protease substrates: Intramolcular dimers described by the excitation model.
Proc Natl Acad Sci USA
93:11640, 1996[Abstract/Free Full Text]
16.
Siegel RM, Martin DA, Zheng L, Ng SY, Bertin J, Cohen J, Lenardo MJ:
Death-effector filaments: Novel cytoplasmic structures that recruit caspases and trigger apoptosis.
J Cell Biol
141:1243, 1998[Abstract/Free Full Text]
17.
Kitabayashi A, Hirokawa M, Hatano Y, Lee M, Kuroki J, Miura AB:
Granulocyte colony-stimulating factor down-regulates allogeneic immune responses by post-transcriptional inhibition of tumor necrosis factor- production.
Blood
86:2220, 1995[Abstract/Free Full Text]
18.
Zamzami N, Susin AS, Marchetti P, Hirsch T, Gomes-Monterrey I, Castedo M, Kroemer G:
Mitochondrial control of nuclear apoptosis.
J Exp Med
183:1533, 1996[Abstract/Free Full Text]
19.
Miura M, Zhu H, Rotello R, Hartwieg EA, Yuan J:
Induction of apoptosis in fibroblasts by IL-1-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3.
Cell
75:653, 1993[Medline]
[Order article via Infotrieve]
20.
Nicholson D, Ali A, Thornberry N, Vaillancourt J, Ding C, Gallant M, Gareau F, Griffin P, Labelle M, Lazebnik Y, Munday N, Raju S, Smulson M, Yamin T, Yu V, Miller D:
Identification and inhibition of ICE/CED-3 protease necessary for mammalian apoptosis.
Nature
376:37, 1995[Medline]
[Order article via Infotrieve]
21.
Enari M, Talanian RV, Wong WW, Nagata S:
Sequential activation of ICE-like and CPP32-like proteases during Fas-mediated apoptosis.
Nature
380:723, 1996[Medline]
[Order article via Infotrieve]
22.
Ohta T, Kinoshita T, Naito M, Nozaki T, Masutani M, Tsuruo T, Miyajima A:
Requirement of the caspase-3/CPP32 protease cascade for apoptotic death following cytokine deprivation in hematopoietic cells.
J Biol Chem
272:23111, 1997[Abstract/Free Full Text]
23.
Susin SA, Zamzami N, Marchetti P, Hirsch T, Gomes-Monterrey I, Castedo M, Kroemer G:
Bcl-2 inhibits the mitochondrial release of an apoptogenic protease.
J Exp Med
184:1331, 1996[Abstract/Free Full Text]
24.
Susin SA, Zamzami N, Marchetti P, Hirsch T, Gomes-Monterrey I, Castedo M, Kroemer G:
Multiple connections between protease activation and mitochondria in Fas/Apo-1/CD95- and ceramide-induced apoptosis.
J Exp Med
186:25, 1997[Abstract/Free Full Text]
25.
Shimizu S, Eguchi Y, Kamiike W, Matsuda H, Tsujimoto Y:
Bcl-2 expression prevents activation of the ICE protease cascade.
Oncogene
12:2251, 1996[Medline]
[Order article via Infotrieve]
26.
Henkart PA:
ICE family protease: Mediators of all apoptotic cell death.
Immunity
4:195, 1996[Medline]
[Order article via Infotrieve]
27.
Martin S, Green D:
Protease activation during apoptosis: Death by a thousand cuts?
Cell
82:349, 1995[Medline]
[Order article via Infotrieve]
28.
Liu X, Zou H, Slaughter C, Wang X:
DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis.
Cell
89:175, 1997[Medline]
[Order article via Infotrieve]
29.
Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X:
Cytochrome C and dATP-dependent formation of apaf-1/caspase-9 complex initiates an apoptotic protease cascade.
Cell
91:479, 1997[Medline]
[Order article via Infotrieve]
30.
Yang J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J, Peng T, Jones DP, Wang X:
Prevention of apoptosis by Bcl-2: Release of cytochrome c from mitochondria blocked.
Science
275:1129, 1997[Abstract/Free Full Text]
31.
Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD:
The release of cytochrome c from mitochondria: A primary site for Bcl-2 regulation of apoptosis.
Science
275:1132, 1997[Abstract/Free Full Text]
32.
Petit PX, Lecoeur H, Zorn E, Dauguet C, Mignotte B, Gougeon ML:
Alterations in mitochondrial structure and function are early events of dexamethasone-induced thymocyte apoptosis.
J Cell Biol
130:157, 1995[Abstract/Free Full Text]
33.
Cossarizza A, Franceschi C, Monti D, Salvioli S, Bellesia E, Rivabene R, Biondo L, Rainaldi G, Tinari A, Malorni W:
Protective effect of N-acetylcysteine in tumor necrosis factor-alpha-induced apoptosis in U937 cells: The role of mitochondria.
Exp Cell Res
220:232, 1995[Medline]
[Order article via Infotrieve]
34.
Finstad HS, Myhrstad MC, Heimli H, Lomo J, Blomhoff HK, Kolset SO, Drevon CA:
Multiplication and death-type of leukemia cell lines exposed to very long-chain polyunsaturated fatty acids.
Leukemia
12:921, 1998[Medline]
[Order article via Infotrieve]
35.
Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A, Nagata S:
A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD.
Nature
391:43, 1998[Medline]
[Order article via Infotrieve]
36.
Sakahira H, Enari M, Nagata S:
Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis.
Nature
391:96, 1998[Medline]
[Order article via Infotrieve]
37.
Liu X, Li P, Widlak P, Zou H, Luo H, Garrard WT, Wang X:
The 40-kDa subunit of DNA fragmentation factor induces DNA fragmentation and chromatin condensation during apoptosis.
Proc Natl Acad Sci USA
95:8461, 1998[Abstract/Free Full Text]
38.
Lowe SW, Ruley HE, Jacks T, Housman DE:
p53-dependent apoptosis modulates the cytotoxity of anticancer agents.
Cell
74:957, 1993[Medline]
[Order article via Infotrieve]
39.
Korsmeyer SJ:
Bcl-2: A repressor of lymphocyte death.
Immunol Today
13:285, 1992[Medline]
[Order article via Infotrieve]
40.
Levine AJ, Momand J, Finlay CA:
The p53 tumour suppressor gene.
Nature
351:453, 1991[Medline]
[Order article via Infotrieve]
41.
Mosmann T:
Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxic assays.
J Immunol Methods
65:55, 1983[Medline]
[Order article via Infotrieve]
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