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(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{dagger}, Akira Yuo{ddagger}, Kiyohiko Hatake§, Fumimaro Takaku§ and Yukihito Ishizaka*

Departments of
* Intractable Diseases and
{ddagger} Hematology, International Medical Center of Japan, Tokyo;
{dagger} Department of Neurology, Neurological Institute, Tokyo Women’s 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
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HL-60 cells undergo apoptosis when placed at room temperature (RT) [Shimura et al. (1997) FEBS Lett. 417, 379–384]. 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 15–25 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
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis, or programmed cell death, is caused by a variety of external or internal stimuli such as {alpha}FAS/tumor necrosis factor {alpha} (TNF-{alpha}) [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 {alpha}FAS [1 ], TNF-{alpha} [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
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
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 {alpha}-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 manufacturer’s 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 ({Delta}{psi}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
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
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 2–6) 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.

{Delta}{psi}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 ({Delta}{psi}m) [40 , 41 ]. As shown in Figure 5 (left panel), {Delta}{psi}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 {Delta}{psi}m was completely blocked (the peak indicated by the solid area). Furthermore, YVAD-CMK also inhibited the decrease of {Delta}{psi}m (Fig. 5 , right panel, compare the peaks shown by the solid line and the solid area). These data suggested that the decrease of {Delta}{psi}m was induced under the control of increased oxidative stress as well as activated ICE-like protease.



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  		Figure 5. Permeability transition of mitochondrial membrane after RT treatment. Mitochondrial membrane potential was studied by using rhodamine 123. Left and right panels showed the effects of NAC and YVAD-CMK on the {Delta}{psi}m, respectively. Solid and dotted lines indicate the membrane potentials of RT-treated cells and control cells, respectively. Effects on the membrane potentials of RT-treated cells in the presence of NAC or YVAD-CMK are shown as solid areas. Addition of both agents inhibited the reduced {Delta}{psi}m caused by RT treatment (compare the peaks shown by a solid line and by solid area).

 
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).



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  		Figure 6. Cytochrome c release from mitochondria by RT treatment. (A) Western blot analysis on cytochrome c. Samples of control cells (lane 1) and RT-treated cells (lane 2) are shown. The addition of CsA greatly inhibited cytochrome c release (lane 3). The molecular masses of each standard proteins are indicated in kilodaltons. (B) Inhibitory effects of CsA on RT-induced apoptosis. FACS analysis on control cells cultured at 37°C (panel 1), RT-treated cells (panel 2), RT-treated cells with DMSO (panel 3), or RT-treated cells with CsA (panel 4) are shown. (C) Effects of CsA on ROS production. ROS was measured on the cells treated at RT in the presence of CsA. Note that the addition of CsA dramatically increased ROS production to about 104-fold (filled squares). ROS production of cells at 37°C (filled circles) and RT (open squares) are also shown. (D) Effects of CsA on the mitochondrial membrane potential. {Delta}{psi}m of cells treated at RT in the presence of CsA was analyzed. Solid and dotted lines indicate {Delta}{psi}m of RT-treated cells and control cells cultured at 37°C, respectively. RT-treated cells with CsA showed the remarkably reduced {Delta}{psi}m described by the solid area.

 
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 {Delta}{psi}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 {Delta}{psi}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 {Delta}{psi}m was exacerbated after 13 h of exposure at RT compared with that observed in 2–8 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 {Delta}{psi}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 1–3) 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
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
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 {Delta}{psi}m. These data clearly indicated that increased oxidative stress was required as one necessary factor. Furthermore, the reduction of {Delta}{psi}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 {Delta}{psi}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.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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