(Journal of Leukocyte Biology. 2000;68:87-96.)
© 2000
by Society for Leukocyte Biology
Oxidative stress as a necessary factor in room temperature-induced apoptosis of HL-60 cells
Mari Shimura*,
Yutaka Osawa
,
Akira Yuo
,
Kiyohiko Hatake
,
Fumimaro Takaku
and
Yukihito Ishizaka*
Departments of
* Intractable Diseases and
Hematology, International Medical Center of Japan, Tokyo;
Department of Neurology, Neurological Institute, Tokyo Womens Medical University, Tokyo; and
Jichi Medical School, Tochigi, Japan
Correspondence: Yukihito Ishizaka, Dept. of Intractable Diseases, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan. E-mail: zakay{at}ri.imcj.go.jp
 |
ABSTRACT
|
|---|
HL-60 cells undergo apoptosis when placed at room temperature (RT)
[Shimura et al. (1997) FEBS Lett. 417, 379384]. We report that
superoxide anion radical, one of the reactive oxygen species (ROS), was
produced after RT treatment. Affinity blot analysis with a biotinylated
YVAD-CHO detected the generation of processed peptides with molecular
masses of 1525 kDa. Activation of such an ICE-like protease was
completely abolished by N-acetylcysteine and exogenously
expressed Bcl-2, known as antioxidants. We concluded that oxidative
stress was a critical factor in the signal cascade of the apoptosis.
Western blot analysis and experiments using tetrapeptide inhibitors
suggested that caspases-1, -3, -4, -6, and -9 did not have an essential
role in the apoptotic cascade. It is interesting that cyclosporin A
(CsA) blocked RT-induced apoptosis with an inhibition of cytochrome
c release from mitochondria. CsA, however, generated a
significant amount of ROS with considerable reduction of mitochondrial
membrane potential, implying that oxidative stress was one necessary
factor for RT-induced apoptosis. It is also likely that mitochondrial
membrane potential and the release of apoptotic factors from cytoplasm
are differently regulated. Taken together with the reports that some
Burkitt lymphoma cells showed apoptosis when exposed at low temperature
followed by rewarming, and that hepatocytes or liver endothelial cells
are susceptible to cold-induced apoptosis through the ROS function, we
propose that studying the mechanism of RT-induced apoptosis of HL-60
cells may provide a therapeutic strategy for pathological conditions
involving ROS, such as neurodegenerative diseases and ischemia.
Key Words: reactive oxygen species YVAD-tagged peptide permeability transition
 |
INTRODUCTION
|
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Apoptosis, or programmed cell death, is caused by a variety of
external or internal stimuli such as
FAS/tumor necrosis factor
(TNF-
) [1
, 2
], anticancer drugs
[3
4
5
], ultraviolet (UV) irradiation [5
],
inhibitors of macromolecule synthesis [6
,
7
], and topoisomerase inhibitors [8
].
Apoptotic signals triggered by these factors provoke dramatic changes
in intracellular effector molecules. These include the activation of
apoptosis-related proteases (caspase) [9
,
10
], mobilization of cytochrome c to cytoplasm
from mitochondria leading to caspase-3 activation [11
,
12
], up-regulation of DNase [13
], and
degradation of death substrates [14
]. Now it is
speculated that the release of cytochrome c to cytoplasm is
caused by disruption of the mitochondrial outer membrane due to an
opening of a permeability transition (PT) pore [15
].
Petronilli et al. reported that the PT pore opening was controlled by
intracellular oxidative stress [16
].
As possible influences on the intracellular oxidative state, reactive
oxygen species (ROS) are thought to have important roles in apoptosis
[17
18
19
20
21
22
]. Antioxidant agents or genes, for example,
N-acetylcysteine (NAC) or Bcl-2, efficiently blocked
apoptosis induced by
FAS [1
], TNF-
[2
], withdrawal of growth factor [23
],
and by HIV [17
]. In addition to these antioxidants,
catalase or superoxide dismutase (SOD) are often used for investigating
the involvement of ROS in apoptosis. For example, apoptosis caused by
peroxynitrite or UV irradiation was efficiently inhibited by SOD
[5
, 19
]. On the other hand, apoptosis
initiated by cytotoxic agents was blocked by catalase
[5
]. There are, however, conflicting findings suggesting
that ROS production is not a critical factor in apoptosis
[24
, 25
]. Jacobson has proposed that there
are two types of apoptosis: one definitely requires an oxidative stress
as a critical factor, and the other works as an acceleration of
apoptotic phenotype [26
].
Recently, we found that cells of HL-60, a human myelogenous leukemia
cell line, underwent apoptosis simply when placed at room temperature
(RT; 21°C) [27
]. Such a phenotype was unique for HL-60
cells because the other 16 human cell lines did not respond to RT
treatment [28
]. A tetrapeptide inhibitor to ICE-like
protease (YVAD-CMK) efficiently blocked the apoptosis, but that to
caspase-3 (DEVD-CHO) did not [28
]. The processing of
caspase-3 was inhibited by YVAD-CMK, indicating that caspase-3
activation, in spite of a downstream event of ICE-like protease
[28
], was dispensable in RT-induced apoptosis.
It is crucially important to note that some kinds of Burkitt lymphoma
cells undergo apoptosis after exposure to cold temperatures at 4 or
25°C [29
]. Furthermore, it was recently reported that
hepatocytes and liver endothelial cells underwent apoptosis at low
temperatures [30
]. Although the precise mechanism of
cold-induced apoptosis of Burkitt cells remains unclear, experimental
data indicated that ROS worked as a causative factor for cold-induced
apoptosis in hepatocytes. Here, we studied the molecular mechanism of
RT-induced apoptosis of HL-60 cells, and found that ROS production was
a critical event. Based on our experimental data, we propose that ROS
production caused ICE-like protease activation, leading to a reduction
of mitochondrial membrane potential. RT-induced apoptosis of HL-60
cells, as a simple system, would provide valuable information on the
molecular pathogenesis of ischemia-induced cell damages
[31
] or for a novel therapeutic strategy of some
leukemias.
 |
MATERIALS AND METHODS
|
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Cell lines and establishment of Bcl-2 transfectants
HL-60 cells were maintained in RPMI 1640 supplemented with 10%
fetal calf serum (FCS) (BioWhittaker, Walkersville, MD). Expression
plasmid of Bcl-2 was provided by Dr. Nuñetz (The University of
Michigan Medical School) [32
]. Thirty micrograms of the
plasmid DNA was digested with BstXI, then transduced into
107 HL-60 cells with a Gene Pulser II (BIO-RAD, Hercules,
CA). The conditions were at 300 volts with 500 µF in 10 mM phosphate
buffer (pH 7.5) and 150 mM NaCl (PBS). Transfectants were selected with
400 µg/mL of G418 (Wako Pure Chemicals, Osaka, Japan). Pooled
transfectants of HL-60 cells introduced with a plasmid expressing
Bcl-xL or a control plasmid were provided by Dr. Kapil
Bhalla (Emory University School of Medicine) [33
]. These
cells were maintained in RPMI 1640 with 10% FCS with G418.
Chemicals
A general inhibitor to apoptosis,
N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone
(zVAD-FMK; Peptide Institute, Osaka), was used at 20 µM as a final
concentration [34
]. Tetrapeptide inhibitors to
caspases-1 (YVAD-CMK;Tyr-Val-Ala-Asp-chroromethylketone), -3
(DEVD-CHO;Asp-Glu-Val-Asp-formaldehyde), -6
(VEID-CHO;Val-Glu-ILe-Asp-formaldehyde), and -9 (LEHD-CHO;
Leu-Glu-His-Asp-formaldehyde), were purchased from the Peptide
Institute. Anisomycin (Sigma, St. Louis, MO), a positive control that
induced apoptosis of HL-60 cells with the processing of caspase-3, was
used at the final concentration of 1 µg/mL [7
]. SOD
and catalase (Sigma) were added at final concentrations of 400 and 800
units/mL, respectively. Ten to fifty millimolar NAC (Wako Pure
Chemicals) was used for studying inhibitory effects on apoptosis. CsA
(provided by Novartis, Japan) was dissolved in dimethyl sulfoxide
(DMSO; Wako) and was added to the culture at 75 µM.
Western blot analysis
Antibodies to caspase-3 (Transduction Lab., Lexington, KY),
caspase-1 (Santa Cruz, Santa Cruz, CA), caspase-1x-4 (MBL,
Gunma, Japan), -6 (PharMingen, San Diego, CA), Bcl-2 (Transduction
Lab.) and cytochrome c (PharMingen) were used as first
antibodies. Anti-mouse IgG (Promega, Madison, WI) and anti-rabbit IgG
(Zymed Laboratory, South San Francisco, CA), conjugated with alkaline
phosphatase, were used as second antibodies. A protein concentration of
each sample was measured by the BCA system (Pierce, Rockford, IL). As
an internal control, antibody to
-tubulin (Sigma) was added
to the solution of the first antibody to cytochrome c.
Immune complex was visualized by 5-bromo-4-chloro-3-indolyl-phosphate
and nitroblue tetrazolium (Sigma) as a substrate.
Detection of apoptotic cells and studying the effects of compounds
on RT-induced apoptosis
FACS analysis of apoptosis was performed by FACScalibur (Becton
Dickinson, San Jose, CA), as described [27
,
28
]. Briefly, after exposure to RT for indicated periods,
HL-60 cells were washed once with PBS and fixed in ice-cold 70%
ethanol. After treatment with 100 µg/mL of RNase A (Sigma), cells
were stained with 50 µg/mL of propidium iodide (Sigma). Then the
population in the hypoploidy region (sub-G1) was calculated by
CELLQuest (Becton Dickinson), and was represented as a percentage.
Detection of annexin V [35
] was performed according to
the manufacturers protocol (MBL). Cells were washed with PBS, then
incubated in a solution of annexin V binding protein. Just before FACS
analysis, cells were incubated in a solution of propidium iodide. By
this procedure, non-necrotic cells that were positive for annexin V
could be identified.
Measurement of superoxide anion radical
Superoxide anion radical was measured by the reported method
[36
]. Cells in batches of about 107 treated
at RT were washed once, then resuspended in Krebs buffer (118 mM NaCl,
4.7 mM KCl, 1.5 mM CaCl2, 25 mM NaHCO3, 1.1 mM
MgSO4, 1.2 mM KH2PO4, with pH 7.4)
[29
]. Each 100-µL solution containing 107
cells was washed once with PBS, then added to 96-multiwell plates
(Packard, Meriden, CT). Lucigenin (Molecular Probes, Eugene, OR) was
added to each well to give 250 mM as a final concentration. Then the
intensity of fluorescence was counted by a microplate scintillation
counter (Packard) at indicated periods up to 60 min after addition of
lucigenin.
Cytochrome c release from mitochondria
Cytoplasmic cytochrome c released from mitochondria
was studied by Western blot analysis [34
]. Cells were
washed once in ice-cold PBS, and then resuspended in the buffer [20 mM
HEPES-KOH, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1
mM dithiothreitol (DTT), 250 mM sucrose, pH 7.5, 0.1 mM] added with
proteinase inhibitors of 0.1 mM phenylmethylsulfonyl fluoride (Wako),
aprotinin (1 µg/mL), leupeptin (1 µg/mL), and pepstatin (1 µg/mL)
(Sigma). Cells were homogenized with a micropestle (Eppendorf, Hamburg,
Germany) on ice. Then the sample was centrifuged twice at 750
g for 10 min at 4°C. The supernatant was recentrifuged
once at 10,000 g for 15 min, followed by ultracentrifugation
at 100,000 g for 30 min by TL-100 (Beckman, Palo Alto, CA).
The supernatant was used as a cytoplasmic fraction. After
centrifugation at 10,000 g, the pellet was used as a
mitochondrial fraction.
Affinity blot analysis using a biotin-conjugated YVAD-CHO
Caspases processed by the RT treatment were analyzed according
to the method described [37
]. Briefly, cell extracts
were prepared by three cycles of freezing and thawing in the lysis
buffer of 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 1 mM PMSF (Sigma),
which contained 20 µM of a biotin-conjugated YVAD-CHO (Calbiochem, La
Jolla, CA). After incubation for 30 min at 37°C, each cell extract
was centrifuged for 20 min at 20,000 g at 4°C, then
applied to 15% SDS-PAGE, followed by blotting to a PVDF membrane
(Nihon Millipore, Tokyo, Japan). Blocking was performed in a solution
of 1% bovine serum albumin with PBS supplemented with 0.02% Tween 20
(PBST). Then the filter was incubated in PBST containing
streptavidin-conjugated alkaline phosphatase (USB) for detecting
peptides tagged with a biotin-conjugated YVAD-CHO.
Analysis of mitochondrial membrane potential
Membrane potential (
m) was measured by using rhodamine 123
(Molecular Probes) [38
]. Briefly, RT-treated HL-60 cells
were further incubated for 30 min in the presence of rhodamine 123 at
the final concentration of 10 µM. After washing three times in PBS,
cells were fixed in ice-cold 70% ethanol, and subjected to FACS
analysis.
 |
RESULTS
|
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RT-induced apoptosis sensitive to a zVAD-FMK inhibitor with an
increased population positive for annexin-V
In our previous report, 300 µM of YVAD-CMK inhibited the
increase of the sub-G1 population after RT treatment
[27
]. In the present study, we first examined the effect
of zVAD-FMK, a general inhibitor to apoptosis, on RT-induced increase
of sub-G1 population. As shown in Figure 1A
, 20 µM of zVAD-FMK completely inhibited any increase in the
sub-G1 population. In addition, RT treatment of HL-60 cells increased
an annexin-V-positive population from 3 to 12%. (Fig. 1B)
[35
]. Taken together with the observation that RT
treatment caused both nuclear condensation and DNA-ladder formation
[27
], we concluded that HL-60 cells underwent apoptosis
by the RT treatment.

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Figure 1. Detection of apoptotic phenotype of HL-60 cells after RT treatment. (A)
Effects of zVAD-FMK on the sub-G1 population after RT treatment.
Control cells cultured at 37°C (panel 1), RT-treated cells without
(panel 2) or with (panel 3) DMSO, and RT-treated cells added with
zVAD-FMK (panel 4) are shown. Note that 20 µM zVAD-FMK completely
blocked RT-induced apoptosis. (B) Increase in annexin V-positive cells
by RT treatment. Populations with a high intensity of FITC detecting
annexin V with standard intensity of propidium (populations in the
lower-right quadrant) were subjected to analysis. Cells showing a high
intensity of propidium iodide were excluded because of necrotic
phenotype [35
]. After 13 h of RT treatment, the
number of cells positive for annexin V increased (right panel). Results
from control cells are also shown (left panel).
|
|
Involvement of ROS production in RT-induced apoptosis and increased
superoxide anion radical
To begin testing the involvement of ROS, we studied the effects of
SOD and catalase, inhibitors to superoxide anion radical and hydroxy
radical. When 400 units/mL of SOD was added to the culture, the sub-G1
population decreased from 60 to 31% (Fig. 2A
, panel 4). On the other hand, catalase was not effective (Fig. 2A
, panel 6). To further confirm that oxidative stress was involved in
RT-induced apoptosis, we looked at the effects of NAC on apoptosis. As
shown in Figure 2B
, the generation of sub-G1 population was completely
abolished by the addition of 50 mM NAC (Fig. 2B
, panel 4). Even 35 mM
NAC could reduce the generation of sub-G1 population (data not shown).

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Figure 2. Effects of reactive oxygen scavengers on RT-induced apoptosis. (A)
Inhibition of apoptosis by SOD. Results of FACS analysis on control
cells cultured at 37°C (panels 1, 3, 5) and RT-treated cells (panels
2, 4, 6). Effects of SOD (panels 3 and 4) or catalase (panels 5 and 6)
on RT-induced apoptosis were studied, respectively. Eight hundred units
per milliliter of SOD inhibited the increase of sub-G1 populations from
60% (panel 2) to 31% (panel 4). (B) Effects of NAC on apoptosis.
Samples shown are control cells cultured at 37°C (panel 1), control
cells added with NAC (panel 2), RT-treated cells (panel 3), and
RT-treated cells with NAC (panel 4). NAC inhibited RT-induced apoptosis
almost completely.
|
|
To obtain direct evidence of ROS production in RT-induced apoptosis,
the generation of superoxide anion radical was measured by using
lucigenin as a substrate [19
]. As shown in Figure 3
, considerable ROS production was observed in the HL-60 cells
exposed to RT for 8 h. When NAC was added to the culture, however,
ROS production was not detected at all (Fig. 3)
.

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Figure 3. Production of superoxide anion radical by RT treatment and its
inhibition by NAC. Superoxide anion radical was measured in cells
cultured at 37°C (open triangles) and cells cultured at 21°C for
8 h (solid circles). ROS production observed at 21°C was
completely blocked by NAC (solid triangles, indicated by arrow).
|
|
Caspase activation as a downstream event of ROS production
Next we characterized the apoptosis-related proteases that showed
affinity to the tetrapeptide motif of substrates of ICE-like protease
[37
]. Because RT-induced apoptosis is sensitive to
YVAD-CMK [27
], we performed an affinity blot analysis
using a biotinylated YVAD-CHO on the extract of apoptotic cells. As
shown in Figure 4A
, RT treatment increased the generation of YVAD-tagged peptides
(Fig. 4A , lane 6). The molecular masses of the detected peptides ranged
from 15 to 25 kDa. Using a mitochondrial fraction, we also detected
such YVAD-tagged peptides (data not shown). Fifty and thirty millimolar
NAC completely inhibited the generation of these peptides (Fig. 4A
,
lanes 2 and 3), suggesting that ROS production was an event upstream of
ICE-like protease activation.

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Figure 4. Involvement of ICE-like proteases in RT-induced apoptosis. (A) Affinity
blot analysis with a biotinylated YVAD-CHO. Peptides showing an
affinity to YVAD-CHO were visualized by using streptavidin-conjugated
alkaline phosphatase. Extracts of cells cultured at 37°C (lane 1) or
at 21°C (panels 26) are shown. Effects of 50 (lane 2), 35 (lane 3),
15 (lane 4), and 10 mM (lane 5) NAC on RT-induced apoptosis are shown.
Numbers on left indicate positions of molecular weight markers. (B)
Western blot analysis of involved caspases. Protein analysis was
carried out by antibodies to caspase-1, -3, -4, and -6. In the results
of caspases-1, -3, and -4, control cells (lane 1), RT-treated cells
(lane 2), and cells with treated anisomycin (indicated by A; lane 3)
were shown. In the results of caspase-6 analysis, control cells (lane
1) and RT-treated cells (lane 2), RT-treated cells with DMSO (lane 3),
or DEVD-CHO (lane 4) and with zVAD-FMK (lane 5) are shown. The
generation of p11 (indicated by arrowhead) was completely blocked by
the addition of zVAD-FMK (lane 5). The positions of molecular weight
markers are indicated on the left. (C) Effects of a tetrapeptide
inhibitor to caspase-6. Samples shown are cells cultured at 37°C
(panel 1), RT-treated cells (panel 2), RT-treated cells with DMSO (lane
3), and RT-treated cells with VEID-CHO (panel 4).
|
|
To characterize the caspases involved in RT-induced apoptosis, Western
blot analysis was performed, and the results are shown in Figure 4B
.
The processing of caspases-3 and -6 was detected, whereas no processing
of caspases-1 and -4 was recognized. Because caspase-6 has been
reported to work downstream of caspase-3 [39
], we
studied the effect of DEVD-CHO on caspase-6 processing, but the result
revealed that caspase-6 activation was independent of caspase-3
activation (Fig. 4B
, lane 4). By contrast, zVAD-FMK completely blocked
caspase-6 processing (Fig. 4B
, lane 5). Next, the effects of a
caspase-6 inhibitor (VEID-CHO) on RT-induced apoptosis was studied. As
shown in Figure 4C
, no inhibitory effects of VEID-CHO on the apoptosis
were observed, suggesting that caspase-6 processing was present, but
dispensable in the apoptotic signal. The caspase responsible for
YVAD-tagged peptides remains to be clarified.

m as the downstream of ROS production and ICE-like protease
activation
Dysfunction of mitochondrial membrane was studied by monitoring
the membrane potential of mitochondria (
m) [40
,
41
]. As shown in Figure 5
(left panel), 
m was decreased in HL-60 cells treated for
6 h at RT (compare peaks indicated by the solid line and the
dotted line for RT-treated and control samples, respectively). By NAC
addition, the RT-induced decrease of 
m was completely blocked
(the peak indicated by the solid area). Furthermore, YVAD-CMK also
inhibited the decrease of 
m (Fig. 5
, right panel, compare the
peaks shown by the solid line and the solid area). These data suggested
that the decrease of 
m was induced under the control of increased
oxidative stress as well as activated ICE-like protease.
Characterization of a CsA-sensitive step inhibiting cytochrome
c release from mitochondria
To clarify the involvement of cytochrome c in
RT-induced apoptosis, cytochrome c release from mitochondria
was studied by Western blot analysis. As shown in Figure 6A
, RT treatment increased the amount of cytoplasmic cytochrome
c (compare lanes 1 and 2).
Because CsA was reported to inhibit the function of the BAX
channel present in the outer membrane of mitochondria
[42
], the effects of CsA on cytochrome c
release and apoptosis were examined. As shown in Figure 6B
, CsA
completely blocked the apoptosis (panel 4). Under the same conditions,
cytochrome c release from mitochondria to cytoplasm was
considerably inhibited (Fig. 6A
, lane 3). We next studied the effects
of CsA on ROS production and the PT of mitochondria. It was surprising
that ROS production was tremendously increased by CsA, up to a
104-fold higher level (Fig. 6C)
. Furthermore, the addition
of CsA remarkably decreased the 
m (Fig. 6D)
.
Kinetics of molecular events in RT-induced apoptosis
The time course of molecular events was analyzed, and the results
are shown in Figure 7
. ROS production was observed in 4 h (Fig. 7A , panel 2),
whereas cytochrome c release from mitochondria to cytoplasm
was also detected in 4 h (Fig. 7B
, lane 3). On the other hand, the
generation of YVAD-tagged peptides and the decrease of 
m were
observed in 2 h after RT treatment (Fig. 7C and 7D)
. It is
interesting to note that there was a unique YVAD-tagged peptide
detected in samples treated for 2 or 4 h at RT (Fig. 7C
,
arrowheads), but it disappeared after 8 h of RT treatment (Fig. 7C
, lane 4). It is also intriguing that the decrease of 
m was
exacerbated after 13 h of exposure at RT compared with that
observed in 28 h (Fig. 7D)
, suggesting two phases of apoptotic
processes, an early and a late phase. At the late phase, mitochondrial
membrane function was completely disrupted after long exposure to RT.

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Figure 7. (A) Time course of the production of superoxide anion radical. ROS was
measured by using lucigenin as a substrate. Results of analysis on
cells treated at RT for 2 (panel 1), 4 (panel 2), 8 (panel 3), and
13 h (panel 4) are shown. Fluorescence intensities elicited from
the cells cultured at 37°C (open squares) or at RT (filled circles),
respectively, are shown. (B) Cytochrome c release from
mitochondria. Control cells (lane 1), RT-treated cells for 2 (lane 2),
4 (lane 3), and 13 h (lane 4) were subjected to analysis. The
positions of standard molecular mass markers are indicated on left. (C)
Generation of YVAD-tagged peptides. Cell extracts of control cells
(lane 1), RT-treated cells for 2 h (lane 2), 4 (lane 3), 8 (lane
4), and 13 h (lane 5) are shown. Two hours after RT treatment,
apoptosis-related YVAD-tagged peptides were already detected (indicated
by an arrowhead in lane 2). Note that such a 25-kDa peptide (lanes 2
and 3) disappeared after 8 h of RT treatment (lane 4). (D)
Mitochondrial membrane potential. The change of  m after RT
treatment is shown. Mitochondrial membrane potentials after 2, 4, and
8 h were plotted by dotted lines. Solid line indicates the data of
cells treated at RT for 13 h. Control sample is described by solid
area.
|
|
Inhibitory effecs of Bcl-2 overexpression on RT-induced apoptosis
Because Bcl-2 is known as an antioxidant
[20
], its effects on RT-induced apoptosis were examined.
First, HL-60 cells were transfected with a plasmid DNA expressing the
Bcl-2 gene, and then a transfectant was obtained (Fig. 8A
). In the Bcl-2 transfectant, exogenous Bcl-2 was overexpressed as
about 26 kDa of protein. In addition, there were bands of 18 and 10
kDa. This observation was also confirmed by using a different
monoclonal antibody purchased from a different company (PharMingen;
data not shown). A previous report by Tsujimoto and Croce has shown
that the Bcl-2 gene, when expressed in vitro,
yielded proteins with different molecular weights [43
].
We speculate that these additional bands are degradation products of
Bcl-2.

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Figure 8. Effects of Bcl-2 overexpression on RT-induced apoptosis. (A) Expression
of exogenous Bcl-2 in HL-60 cells. Control cells with an expression
vector and Bcl-2 transfectant were subjected to Western blot analysis.
Arrows indicate exogenous Bcl-2. (B) Inhibited RT-induced apoptosis in
the Bcl-2 transfectant. RT-induced apoptosis was analyzed on the
control neo transfectant (panels 1, 3) and a Bcl-2
transfectant (panels 2, 4). Results of control samples cultured at
37°C (panels 1 and 2) and cells at 21°C (panels 3 and 4) are shown.
(C) Effects of Bcl-2 on the processing of peptides tagged with a
biotinylated YVAD-CHO. Affinity blot analysis was performed after RT
treatment on control neo transfectant (lanes 13) and Bcl-2
transfectant (lanes 4 and 5). Effects of zVAD-FMK on the processing of
the peptides were also studied (lane 3). In the Bcl-2 transfectant,
processing of the protein showing affinity to YVAD-CHO was completely
abolished (lane 5). zVAD-FMK also completely blocked the generation of
YVAD-tagged peptides (lane 3).
|
|
The effects of RT treatment on such a clone were investigated. As shown
in Figure 8B
, apoptosis was almost completely blocked (panel 4).
Furthermore, affinity blot analysis revealed that no YVAD-tagged
peptides were generated in a Bcl-2 transfectant even after exposure to
RT for 13 h (compare lanes 2 and 4 in Fig. 8C
). Data indicated
that Bcl-2 functioned as the upstream molecule of ICE-like protease. In
addition to Bcl-2, we tested the effects of Bcl-xL on
RT-induced apoptosis, and found that Bcl-xL also inhibited
RT-induced apoptosis (data not shown).
 |
DISCUSSION
|
|---|
In the present study, we found that RT-induced apoptosis of HL-60
cells induced various molecular events such as production of superoxide
anion radical, activation of ICE-like protease, a decrease of
mitochondrial membrane potential, and cytochrome c release
from mitochondria. Among these, increased oxidative stress was proven
critical in the apoptotic cascade because antioxidants such as SOD,
NAC, and Bcl-2 overexpression inhibited apoptosis. Although Jacobson
has raised the possibility that ROS occasionally works as an
accelerator instead of an inducer of apoptosis, our present work
indicates that ROS is crucial for the execution of apoptosis. Because
the processing of ICE-like protease was completely abolished by these
antioxidants (Figs. 4A and 8C)
, we concluded that ROS production,
resulting in the increased oxidative state, was upstream of caspase
activation.
It has been reported that ROS production is related to a variety of
pathological conditions such as neurodegenerative diseases
[44
, 45
] and ischemia [31
].
Recent findings by Rauen et al. indicated that cold-induced apoptosis
found in hepatocytes and liver endothelial cells was mediated by ROS
[30
]. Although the molecular mechanism of cold-induced
apoptosis observed in some Burkitt lymphoma cases remains to be
clarified [29
], it is tempting to speculate that ROS
also plays an important role in these malignant cells. RT-induced
apoptosis of HL-60 cells would yield information on the molecular
mechanism of ROS-induced apoptosis that would provide a rationale for a
novel therapeutic strategy for leukemias as well as other intractable
diseases.
Although cytochrome c release from mitochondria with
caspase-3 activation was detected in RT-induced apoptosis, an inhibitor
to caspase-3 (DEVD-CHO) did not block the apoptosis
[27
]. These data suggested that the cytochrome
c-dependent caspase-3 activation was not crucial in the
signal pathway. We tried to characterize the involved ICE-like protease
by using antibodies to various caspases. Although the processing of
caspase-6 was observed (Fig. 4B)
, its inhibitor (VEID-CHO) did not
block the apoptosis (Fig. 4C)
, suggesting that the processing of
caspase-6 did not have an essential role in RT-induced apoptosis. An
affinity blot analysis on YVAD-tagged peptides revealed the presence of
a unique band with a molecular mass of about 25 kDa that was detected
in the samples treated for 2 and 4 h at RT (Fig. 7C) . It is
interesting to note that it disappeared after 8 h of RT treatment.
Although YVAD-tagged peptides with molecular masses ranging from 17 to
20 kDa are known to be the processed caspase-3 and caspase-6
[46
], there has been no report on the 24-kDa protein. An
uncharacterized protease might be activated at an early phase of
RT-induced apoptosis.
We showed that CsA completely blocked the apoptosis (Fig. 6A
and 6B) with a concomitant inhibition of cytochrome c release from
mitochondria. To our surprise, however, CsA caused a considerable
amount of ROS production with reduced 
m. These data clearly
indicated that increased oxidative stress was required as one necessary
factor. Furthermore, the reduction of 
m and the release of
cytochrome c from mitochondria would be differently
regulated, as reported [41
]. Now the release of
cytochrome c from mitochondria is explained by the
disruption of the mitochondrial outer membrane, which is supposed to be
a result of the expansion of mitochondrial volume caused by PT pore
opening. Our present results clearly showed that the decrease of

m caused by CsA did not directly result in the release of
cytochrome c. Because CsA targets the BAX channel present in
the mitochondrial outer membrane [42
], it is reasonable
to speculate that the BAX channel or its related molecule regulates the
release of cytochrome c in a CsA-sensitive manner. The
release of cytochrome c from mitochondria would not be
simply because of a disruption of the mitochondrial outer membrane.
In the experiments on the kinetics of RT-induced apoptosis, we observed
a generation of YVAD-tagged peptides and the change of mitochondrial
membrane potential after 2 h of RT treatment. On the other hand,
the production of superoxide anion radical and cytochrome c
release from mitochondria were observed after 4 h of the same
treatment. Although there is a time difference between the detection of
YVAD-tagged peptides and ROS production, results of experiments using
NAC and overexpressed Bcl-2 strongly suggested that an increased
oxidative stress was an event upstream of ICE-like protease activation.
It is tempting to speculate that an initial increase of oxidative
stress, which was required for triggering the apoptotic signal, would
be too small to detect by any conventional method.
The initial molecular event is the most important question for
understanding the mechanism of RT-induced apoptosis of HL-60 cells. As
preliminary data, staurosporine, an inhibitor of tyrosine kinase,
inhibited apoptosis [M. Shimura, unpublished results]. Because Bcl-2
loses its anti-apoptotic activity by hyperphosphorylation
[47
], an increase of intracellular phosphorylation state
due to an unbalanced ATP metabolism is one possible explanation for the
mechanism. We are now focusing on the change of mitochondrial function
before and after RT treatment.
 |
ACKNOWLEDGEMENTS
|
|---|
We are grateful to Dr. Kumiko Saeki (International Medical Center
of Japan) for critical advice on this work. We also thank Drs. Gabriel
Nuñez (The University of Michigan Medical School) and Kapil
Bhalle (Emory University School of Medicine) for providing us with
Bcl-2 plasmid and Bcl-xL transfectants of HL-60 cells,
respectively. Cyclosporin A was kindly provided by Novartis. We also
express heartfelt thanks to Dr. Yoshifumi Takeda (Director General,
National Institute of Infectious Diseases, Tokyo) for his support of
our work. We thank Dr. Dovie Wylie for editorial assistance. This work
was supported by a Grant-in-Aid for the Second Term Comprehensive
10-year Strategy for Cancer Control from the Ministry of Health and
Welfare, Japan.
Received August 12, 1999;
revised February 28, 2000;
accepted February 29, 2000.
 |
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