Fusarenon-X induced apoptosis in HL-60 cells depends on caspase activation and cytochrome c release

Toxicology 172 (2002) 103– 112 www.elsevier.com/locate/toxicol

Fusarenon-X induced apoptosis in HL-60 cells depends on caspase activation and cytochrome c release Katsuhiro Miura *, Leila Aminova 1, Yuichi Murayama 2 Department of Safety Research, National Institute of Animal Health, 3 -1 -5 Kannondai, Tsukuba, Ibaraki 305, Japan Received 30 January 2001; received in revised form 27 September 2001; accepted 30 November 2001

Abstract Fusarenon-X (FX), a trichothecene mycotoxin, is well known to be cytotoxic to mammalian cells. Our previous study revealed that FX induced apoptosis in mouse thymocytes both in vivo and in vitro. We investigated the mode of apoptosis induced by FX using HL-60 cell culture. When FX at a final concentration of 0.5 mg/ml was added, cell degradation was observed 5 h after exposure, and most of the cells had fallen into apoptosis 24 h after exposure. DNA fragmentation into 180-bp multimers was observed 5 h after exposure, and its dose-dependency was clear in the cells treated with 0.1 mg/ml and higher doses. The percentage of apoptotic cells (sub-G0 population) increased doseand time-dependently after exposure, when analyzed using flow cytometry. The activities of caspase-3, -8, and -9 were elevated within 2 h by exposure to FX. DNA fragmentation and an increase in the apoptotic population were abrogated by pre-treating the cells with broad-spectrum caspase inhibitors Z-VAD-fmk or Z-Asp-CH2-DCB. Cytochrome c release from mitochondria to cytoplasm was observed clearly, and this release occurred caspase-independently. These findings suggest that FX induces apoptosis in HL-60 cells by stimulating cytochrome c release followed by its downstream events including the activation of multiple caspases. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Apoptosis; Fusarenon-X; Mycotoxin; HL-60; Caspase; Cytochrome c; Flow cytometry

1. Introduction

* Corresponding author. Tel.: + 81-298-38-7813; fax: + 81298-38-7825. E-mail address: [email protected] (K. Miura). 1 Present address: Department of Microbiology, University of Illinois, Goodwin Avenue, Chemical and Life Sciences Building, B103, Urbana, IL 61820, USA. 2 Present address: Japan Science and Technology Corporation.

Apoptosis is an intrinsic cell removal mechanism that occurs during development and in response to various hazardous stimuli. Many xenobiotics such as anti-cancer drugs, bacterial, plant, and fungal toxins, infections, and irradiation trigger cell apoptosis (Pestka et al., 1994; Komatsu et al., 1998; Gougeon and Montagnier, 1999; Radford 1999; Gamen et al., 2000). Tricothec toxins produced by Fusarium fungi cause

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mycotoxicoses in human beings and animals who have ingested food (feed) contaminated with fungi (Tandon et al., 1990) and induce apoptosis in animals and cultured cells (Ueno et al., 1995). Fusarenon-X (FX), a trichothecene mycotoxin, is known to be cytotoxic to many kinds of mammalian cells (Ohotsubo and Saito, 1970; Ueno et al., 1973; Bondy et al., 1991). In a previous report, we indicated that FX is a potent inducer of apoptosis in mouse thymocytes both in vivo and in vitro (Miura et al., 1998). This finding, as well as the evidence of apoptotic changes in rat gastric glandular cells caused by FX (Li and Shimizu, 1997), is consistent with the fact that FX has a strong cytotoxicity to rapidly dividing cells (Saito et al., 1969; Ueno et al., 1971). The cytotoxic effects of trichothecenes are thought to depend on the inhibition of protein synthesis. FX binds to the peptidyltransferase catalytic center on ribosomes and blocks the elongation of the peptide chain (Carter and Cannon, 1978), but the mode of cell death in cells exposed to trichothecenes has so far not been fully understood. In this paper we report that FX induces apoptosis in cultured HL60 cells by promoting cytochrome c release from mitochondria and by activating caspase family proteins.

2. Materials and methods

2.1. Mycotoxin Purified FX (Wako Chemical Co., Tokyo, Japan) was dissolved in dimethylsulfoxide (DMSO) and used as described in our previous report (Miura et al., 1998). The final concentration of DMSO in the FX sample was under 0.1%.

2.2. Cells The human promyelocytic leukemia cell line, HL-60, was supplied from RIKEN Cell Bank (Tsukuba, Japan). RPMI-1640 medium (GIBCO, Invitrogen Corp., Carlsbad, CA, USA) containing 10% newborn calf serum, 40 mg/ml kanamycin sulfate (GIBCO) and 2.5 mg/ml fungizone (GIBCO) was used. Exponentially growing cells

cultured in fresh media for 2–3 days were used for the experiment after we verified that their growth pattern was almost the same as that reported originally (Foa et al., 1982).

2.3. Obser6ation of cell degradation and growth inhibition in HL-60 exposed to FX To determine the effects of FX on the morphology and growth of HL-60 cells, FX was added to the cell culture (5.2× 105 cells/ml) at a final concentration of 0.1, 0.2, or 0.5 mg/ml. Cells were incubated using a 60× 15 mm culture dishes (FALCON, Becton Dickinson, NJ, USA) (final volume 3 ml/dish) at 37 °C in a humidified 5% CO2 atmosphere. Morphological changes were observed after the addition of FX using a phasecontrast inverse microscope (IMT-2, Olympus Co., Ltd, Tokyo, Japan). Viable cell numbers were determined 5, 24, 48, and 72 h after the addition of FX by means of the trypan blue exclusion method using a hemocytometer. Time after the addition of FX is abbreviated as HAF (hours after FX administration).

2.4. Detection of DNA fragmentation HL-60 cells (5×105) were incubated in 500 ml of medium with different concentrations of FX for 5 h using a 24-well culture plates (FALCON). Then cells were harvested, washed with phosphate-buffered saline (PBS) and centrifuged at 3000 rpm. DNA extraction and its analysis with 2% agarose gel electrophoresis were carried out according to the methods described in our previous report (Miura et al., 1998). The DNA size marker used was 100 Base Pair Ladder (Amersham Pharmacia Biotech, Buckinghamshire, England) or Fx174/HaeIII digest (Nippon Gene, Tokyo, Japan).

2.5. Caspase inhibitor The effect of three broad-spectrum caspase inhibitors, Z-Asp-CH2-DCB, Boc-Asp(Obzl)-cmk, and Z-VAD-fmk, on FX-induced apoptosis in HL-60 cells was assessed. These compounds were dissolved in DMSO and diluted with PBS at the

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time of use. For the control experiment, DMSO solution was used at a dilution comparable to that used in the experiments with inhibitors. HL-60 cells were incubated for 1 h either alone or in the presence of inhibitors. They were then incubated further for 4 h in the presence of FX added at a final concentration of 0.5 mg/ml. The effects of inhibitors were then assessed in terms of DNA fragmentation or increment of sub-G0 apoptotic population. Experimental conditions are described with corresponding results (Fig. 4; Table 1). Inhibitors were purchased from the Peptide Institute Inc. (Osaka, Japan), except for BocAsp(Obzl)-cmk (Calbiochem-Novabiochem Corp., CA, USA).

2.6. Assay of caspase acti6ity We tested whether caspase-3, -8 and -9 were activated in HL-60 cells exposed to FX. Cells were cultured in the presence or absence of FX,

Table 1 Effects of caspase inhibitors on the percentage of apoptotic cells among HL-60 cells treated with FX Inducer (dose a)

Inhibitors (dose a)

% of apoptotic population

FX (0.5 mg/ml)

Control (none) b Z-Asp-CH2-DCB (100 mg/ml) Boc-Asp(Obzl)-cmk (5 mM)

19.6 1.3

Control (none) b Z-Asp-CH2-DCB (100 mg/ml) Boc-Asp(Obzl)-cmk (5 mM)

13.1 1.9

Etoposide (10 mM)

1.4

3.9

HL-60 cells (2.5×106) were pre-treated with the indicated concentration of inhibitors for 1 h in 3 ml of media; then 1.5 ml of the inducer listed was added, and the cells were incubated for 5 h. The percentage of apoptotic cells was determined by a flow cytometer, as described in Section 2.7. Data is the average of two samples. a Final concentration. b Control cells treated with only FX. The percentage of apoptotic cells treated with only inhibitors was almost equal to that of the control values without either FX or inhibitors (1–2%).

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then harvested at 2 HAF and 4 or 5 HAF (see Fig. 5). FX-treated and non-treated control cells were lysed, and their caspase activities were determined using colorimetric protease assay kits (BVK106, -K113 and -K119 for caspase-3, -8 and -9, respectively; MBL, Nagoya, Japan), according to the instructions provided by the manufacturer. An equal number of cells were used in each experiment, i.e. 1× 106 cells/sample for caspase-3 and 4.0×105 cells/sample for caspase-8 and -9, so that the cell lysates had adequate protein concentrations (1–4 mg/ml of sample) for measurements. Enzymatic reaction was carried out for 2 h at 37 °C after the addition of caspase-3, -8, and -9-specific tetra-peptide substrates, i.e. DEVDchromophore p-nitroanilide (pNA), IETD-pNA and LEHD-pNA, respectively, using 96-well culture plates (FALCON) in duplicate or triplicate. Calorimetric assays of caspases were carried out by measuring absorbance at 405 nm with a microplate reader (Molecular Devices Corp., Sunnyvale, CA, USA).

2.7. Assessment of apotosis by flow cytometry For the quantitative analysis of apoptosis in FX-treated HL-60 cells, we modified a method (Douglas et al., 1995) to discriminate the smallsized apoptotic cells from debris. Briefly, cells were fixed with 70% ethanol, treated with RNase, stained with propidium iodide and then analyzed with a flow cytometer (Profile 2, Coulter, Tokyo, Japan). The percentage of apoptotic cells in the total cell population was obtained by dividing particle counts in the sub-G0 apoptotic population by the total counts in the sub-G0-, G0/G1-, S- and G2/M-phase populations. We confirmed that this measurement detects cells changed apoptotically, because pre-treatment with the DNase inhibitor ZnCl2 completely suppressed the sub-G0 apoptotic population that appears in cells treated with FX.

2.8. Detection of mitochondrial cytochrome c HL-60 cells were incubated in medium containing 25 mM Z-VAD-fmk for 1 h at 37 °C in a humidified 5% CO2 atmosphere or without Z-

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Fig. 1. Cellular degradation of HL-60 cells exposed to FX. Cells were incubated with (B) or without (A) 1.0 mg/ml of FX for 5 h, after which photomicrographs were taken under phase contrast (200 ×).

VAD-fmk as a control. FX was added to the cultures at a final concentration of 0.5 mg/ml, and the cells were then further incubated for 2 h. The detection of mitochondrial cytochrome c was carried out as reported earlier (Yang et al., 1997). After washing, the cell pellets were resuspended at 5× 107 cell/ml in extraction buffer (20 mM Hepes-pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride and 250 mM sucrose) and homogenized using a micro-homogenizer, S-205 (Ikeda Rika, Tokyo, Japan). The homogenates were centrifuged at 750×g for 10 min at 4 °C. The supernatants were centrifuged at 10000× g for 15 min at 4 °C, and the pellets were resuspended in 50 ml of extraction buffer (designated the mitochondrial fraction). The supernatants separated by a 10000×g spin were further centrifuged at 100000× g for 1 h at 4 °C, and the resulting supernatants were collected (designated the cytosol fraction). Mitochondrial and cytosol fractions were separated by electrophoresis on a 12.5% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and then transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). After blocking, the membrane was incubated with anti-cytochrome c monoclonal antibody (7H8.2C12, Pharmingen, San Diego, CA,

USA) and then with a goat anti-mouse immunoglobulin F(ab%)2 antibody conjugated with peroxidase (BIOsource, Camarillo, CA, USA). Specific bands were visualized by color development.

2.9. Statistics The differences between the activity of caspases in FX-treated and non-treated cells were analyzed by one-factor analysis of variance (ANOVA), performing multiple comparisons by Fisher’s least significant difference test (Fig. 5). Two-factor ANOVA was applied to check the difference in the percentage of apoptotic populations between FX-dose groups or between treatment-time groups, respectively (Section 3.2; Fig. 3). The statistical analysis program STATVIEW (SAS Institute Inc., Cary, NC, USA) was used for the above tests.

3. Results

3.1. Growth inhibition, cell degradation, and DNA fragmentation in HL-60 cells exposed to FX In the experiment carried out to examine the effects of FX on the morphology of HL-60 cells

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(Section 2.3), cell degradation was observed 5 h after exposure (HAF), decreasing viable cell number (Fig. 1) when a 0.5 mg/ml concentration of FX was used. Then, most of the cells were broken into small cellular bodies by 24 HAF. The effects of 0.1 mg/ml of FX on cell proliferation were also clear because viable cell numbers drastically decreased from the initial 5.2× 105 to 3.6×105 cells/ml at 72 HAF accompanied with cell degradation, even though a slight increase in viable cell numbers were observed at 24 and 48 h HAF (6.4×105 and 6.6×105 cells/ml, respectively). An intermediate effect between 0.5 and 0.1 mg/ml was observed in the experiment where 0.2 mg/ml of FX was applied. In contrast with these, the viable cell numbers in the culture without toxin had increased at 24, 48 and 72 HAF (9.2, 14.5 and 16.4×105 cells/ml, respectively), showing normal cell morphology. Induction of DNA fragmentation by FX were examined (Section 2.4). As shown in Fig. 2, DNA fragmentation by 180-bp multimers (ladder) was observed in the cells exposed to 0.1 mg/ml of FX for 5 h. The DNA ladder patterns were dose-dependently clearer in the samples treated with 0.2–0.8 mg/ml of FX. To determine the

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Fig. 3. Dose- and time-dependent increase of apoptotic population in HL-60 cells treated with FX. 2.5 × 106 cells in 4.5 ml of medium were incubated with the indicated concentration of FX at the start. A total of 2 × 104 cells were analyzed at 5, 24, 48, 72 and 96 HAF using a flow cytometer to determine sub-G0 percentages by the method described in Section 2.7. UC indicates that the sub-G0 apoptotic population grew predominant over the cell cycle histograms and were inseparable from G0/G1-, S-, and G2/M-peak at the indicated times. Analyses were performed twice for each sample. Data represent the results of two similar experiments. Plotted marks indicate means and standard deviations.

minimum exposure time after which HL-60 cells undergo apoptotis, we replaced the FX containing culture media with fresh ones at 5, 10, 20, and 30 min after treatment with 0.5 mg/ml of FX. DNA fragmentation in the cells was then checked 5 h later by means of gel electrophoresis. No DNA ladder patterns or only trace patterns were seen in the cells exposed to FX for 5 or 10 min, respectively; however, clear DNA ladder patterns were observed in the cells exposed for 20 min or longer (data not shown). Thus, it was shown that cells proceed to an irreversible step of apoptosis within 20 min.

3.2. Time- and dose-dependent increase of apoptotic cell population in FX-treated HL-60 cells

Fig. 2. Induction of DNA fragmentation in HL-60 cells exposed to FX. HL-60 cells were incubated with the indicated concentrations of FX for 5 h and were then harvested. Genomic DNAs were extracted and underwent 2% agarose gel electrophoresis. Representative data were obtained in two experiments at each concentration. M: 100 base ladder DNA marker. C: Control sample incubated with medium only.

We investigated the time course of the increment of the apoptotic cells in FX-treated HL-60 cells, quantifying the apoptotic percentage of the sub-G0 population using the method described in Section 2.7. As shown in Fig. 3, the addition of 0.5 mg/ml FX increased the percentage of apoptotic cells to 21.8% at 5 HAF and then to 41.4% at 24 HAF. At 48 HAF, the sub-G0 apoptotic cell population became predominant, covering the en-

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tire area of the histogram. Treatments with 0.2 and 0.1 mg/ml of FX also induced increments of apoptotic cells, that is, 54.1 and 33.3% at 48 HAF, respectively. The apoptotic cell population became predominant at 72 and 96 HAF in the case of 0.2 and 0.1 mg/ml FX treatment, respectively. When the difference of apoptosis percentage between three FX-dose groups (0, 0.1 and 0.2 mg/ml) and that between four FX-exposure time groups (0, 5, 24 and 48 HAF) were analyzed using two-factor ANOVA, significant differences (P B 0.001) were observed in both comparisons. This indicates that apoptotic changes induced by FX depend both on the exposure dose and time in HL-60 cells.

3.3. Effects of caspase inhibitors on DNA fragmentation and apoptotic changes in FX-treated HL-60 cells To examine whether caspase activation cascades were involved in this apoptotic cell death, we tested the effects of caspase inhibitors on DNA fragmentation and apoptotic changes in HL-60 cells exposed to FX. When we pre-treated a total 2× 105 HL-60 cells with a broad-spectrum cell-permeable caspase inhibitor, Z-VAD-fmk or Z-Asp-CH2-DCB, both inhibitors suppressed strongly the DNA fragmentation induced by 0.5 mg/ml of FX (Fig. 4). Cellular degradations observed microscopically in cultures incubated with 0.5 mg/ml of FX were totally abrogated in the culture pre-incubated with Z-VAD-fmk or Z-AspCH2-DCB and then treated with this concentration of FX (data not shown). This was confirmed, using flow cytometry, in terms of the abrogation of the degraded apoptotic cell (sub-G0) population in FX-treated HL-60 cells that were pre-treated with Z-Asp-CH2-DCB and BocAsp(Obzl)-cmk (Table 1). The same situations were seen when etoposide, a chemical inducer of apoptosis, was administrated to HL-60 cells.

3.4. Acti6ation of caspase in the HL-60 cells treated with FX To investigate whether initiator caspases (caspase-8, -9) and an effector caspase (caspase-3) are

involved in the FX-induced apoptosis, we measured the activities of these caspases in HL-60 cell lysates after treatment with FX. As shown in Fig. 5, the activities of these three caspases were remarkably elevated 2 h after the addition of 0.5 mg/ml of FX and almost equal activities were retained for 3 h (caspase-3, -8) or 2 h (caspase-9) thereafter.

3.5. Cytochrome c release from mitochondria to cytoplasm in the HL-60 cells treated with FX As shown in Fig. 6, cytochrome c was detected mainly in the cytosol fraction in the FX-treated HL-60 cells, indicating that cytochrome c was released from mitochondria to cytoplasm by FX stimulation (Fig. 6(a) and (b)). Pre-treatment with a caspase inhibitor, Z-VAD-fmk, effectively inhibited DNA fragmentation following the treatment with FX (data not shown). However, most cytochrome c was located in the cytoplasm of these

Fig. 4. Caspase inhibitors block DNA fragmentation in HL-60 cells treated with FX. 2 ×105 cells were cultured in 1 ml in the presence of 100 mg/ml Z-Asp-CH2-DCB (ZD) or 80 mM Z-VAD-fmk (ZV) for 1 h using 24-well plate (FALCON). The cell cultures added 5 ml of solution containing FX at 0.1 mg/ml were further incubated for 4 h. DNA samples were analyzed by means of 2% agarose gel electrophoresis. Cell cultures with or without inhibitors contain 1% (lanes 1 – 4) or 0.2% (lanes 6 – 9) DMSO. DNA marker (F× 174/HaeIII digest) was applied to lane 5. The arrow indicates the band of 234 bp.

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Fig. 5. Activation of caspase-3, -8, and -9 in HL-60 cells treated with FX. Cells were cultured with FX for the indicated times. Caspase activities in the lysates from FX-treated and untreated (CONT) cells were measured. The histogram shows means and standard deviations. Samples were obtained from duplicated (caspase-3, -8) or triplicated cultures (caspase-9). Shown are the representative results of two independent experiments for each caspase. Values marked with a different superscript letters (a, b) are significantly different from one another (caspase-3: PB 0.001; caspase-8: PB 0.05; caspase-9: PB0.001).

cells (Fig. 6(c)), suggesting that cytochrome c release from mitochondria induced by FX could not be suppressed by the Z-VAD-fmk treatment.

4. Discussion We have reported earlier that FX, a trichothecene mycotoxin, is capable of inducing apoptosis in mouse thymocytes both in vitro and in vivo (Miura et al., 1998). In the present study, we demonstrated that a decrease in cell viability, cell degradation to apoptotic bodies and internucleosomal fragmentation of DNA were vigorously induced in human promyelocytic myeloma HL-60 cells exposed to FX. DNA fragmentation was detected at 5 HAF with exposure to 0.5 mg/ml FX (Section 3.1). The decrease in cell viability and apoptotic body formation (sub-G0 peak shown by flow cytometry) proceeded time- and dose-dependently (Section 3.2). Twenty minutes of treatment was enough to induce DNA fragmentation in the cells (Section 3.1). This indicates that the apoptotic event is triggered as early as 20 min after exposure to FX, and that thereafter the apoptotic signal is transduced to an irreversible step. Ohotsubo et al. (1972) had reported that the treatment of mouse L-cells with 2– 3 mg/ml of FX for 5 min was sufficient to induce polyribosomal break-

down, i.e. protein synthesis inhibition. There seems to be some relationship between rapid ribosomal breakdown and early triggering of apoptosis. DNA fragmentation or apoptotic body formation in FX-treated HL-60 cells were efficiently inhibited with broad-spectrum caspase inhibitors (Section 3.3), and caspase-8 and -9 (initiator caspases) and caspase-3 (an effector caspase) were markedly activated (Section 3.4). These findings indicate that a caspase-activating cascade is involved in FX-induced apoptosis.

Fig. 6. Cytochrome c release from mitochondria in the HL-60 cells treated with FX. HL-60 cells were preincubated with (c) or without (a, b) caspase inhibitor Z-VAD-fmk (25 mM) for 1 h, and they were then treated with 0.5 mg/ml FX for 2 h. Mitochondria (M) and cytosol (C) fraction were prepared and analyzed as described in Section 2.8. The representative results of the three experiments are shown.

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There are two main pathways for the initiation of a caspase cascade that eventually leads to cell death by apoptosis. One is the pathway mediated by cell surface receptors such as Fas/TNF receptor I. In this pathway, receptor-associated adapter protein FADD (Fas-associated protein with death domain) activates pro-caspase-8, and active caspase-8 activates multiple caspases (Boldin et al., 1996; Vincenz and Dixit, 1997). The other pathway is triggered by mitochondrial cytochrome c release to cytoplasm. Cytochrome c, in association with ATP, binds to Apaf-1 (apoptotic protease activating factor-1), enabling this protein to recruit and activate caspase-9, leading to caspase-3 and other caspase activation events (Sun et al., 1999; Slee et al., 2000). Recently, we reported that when human Fas cDNAs were transfected into Fas-negative K562 cells, Fas-positive transfectants became apoptosissensitive to anti-Fas monoclonal antibodies (CH11) and Fas ligands (Murayama et al., 2000). However, there were no differences between Faspositive and -negative K562 cells in sensitivity to FX-mediated apoptosis (Murayama, data not shown). These observations suggest that FX does not transmit a death signal as a part of the cell death mechanism that engages the Fas/FADDmediated pathway. In the present study, we showed that cytochrome c was released from mitochondria to cytosol after the FX-treatment in HL-60 cells (Fig. 6(b)). This translocation of cytochrome c occurred on condition that Z-VAD-fmk almost entirely suppresses DNA fragmentation in FXtreated cells (Fig. 6(c)). Therefore, it is suggested that Z-VAD-fmk inhibits the caspase cascade reaction downstream of the cytochrome c release event, and that cytochrome c release itself does not depend on caspase activation. These findings indicate that the caspase-independent release of mitochondrial cytochrome c is a crucial trigger in FX-induced apoptosis, as demonstrated in other physico-chemically induced apoptoses such as etoposide and staurosporine, and by UV irradiation (Bossy-Wetzel et al., 1998; Zhuang et al., 1998). Once a small amount of cytochrome c is released, Apaf-1-mediated activation of caspase-9 occurs, followed by the activation of caspase-3

and -8 and the cleavage of Bid (a BH-3 domaincontaining Bcl-2 family protein) that amplifies cytochrome c release (Slee et al., 1999, 2000). The present data suggested that cytochrome c release occurs as the initial step of FX-mediated apoptosis in HL-60 cells, provoking the downstream amplification loop, with all these events finally leading to apoptotic cell death. It is invaluable to find mechanisms by which cytochrome c translocates from death-committed mitochondria to cytoplasm in a caspase-independent manner in chemically induced apoptosis. Recently, Li et al. (2000) found that TR3 (nur77), a steroid–thyroid hormone–retinoid receptor operating as a nuclear transcription factor, translocates from the nucleus to the mitochondria and then induces cytochrome c release, followed by apoptosis, in response to etoposide or other chemical factors. It has been reported that trichothecene mycotoxins induce apoptosis (Pestka et al., 1994; Ueno et al., 1995; Ihara et al., 1997) and, at the same time, they, including FX, are effective inhibitors of protein synthesis (Ohotsubo et al., 1972; Ueno et al., 1973; Cundliffe et al., 1974; Carter and Cannon, 1978). The relationship between trichothecene-induced apoptosis and mitogen-activated protein kinase activation was studied recently (Shifrin and Anderson, 1999; Yang et al., 2000). Shifrin and Anderson (1999) reported that selected trichothecenes activate c-Jun N-terminal kinase (JNK)/p38 mitogen-activated kinase and induce apoptosis (i.e. internucleosomal DNA fragmentation and caspase-3 activation) in Jurkat cells. They suggested that the binding of trichothecene to the peptidyltransferase site on the ribosomes initiated both JNK/p38 kinase activation and apoptosis. However, the mechanisms by which apoptosis is induced through these functional effects of trichothecene remain to be elucidated. It was suggested that the disruption of mitochondrial transmembrane potential and activation of caspase-3 were provoked by trichothecene (T-2 toxin) (Okumura et al., 1998). An outline of FX-induced apoptosis can be drawn based upon our present study and the above-mentioned knowledge on trichothecene toxicity. However, the upstream trigger factor(s) that induce mitochondrial cytochrome c translocation in the

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cell death caused by fusarium mycotoxin should be investigated further. This may advance the development of methods for the prevention of toxicoses due to trichothecene mycotoxin in humans and animals.

Acknowledgements The authors thank Drs Yasuyuki Nakajima, Yoshiko Motoi, and Nobuyuki Terakado of the National Institute of Animal Health, Tsukuba, for their encouragement, and Dr Nobuo Goto for his helpful advice on the manuscript. We also thank Mr Tasuku Yamashita of the Japan Science and Technology Corporation and Mrs Kaori Saitoh of the National Institute of Animal Health, Tsukuba, for their skillful technical assistance.

References Boldin, M.P., Goncharov, T.M., Goltsev, Y.V., Wallach, D., 1996. Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell 85, 803 –815. Bondy, G.S., McCormick, S.P., Beremand, M.N., Pestka, J.J., 1991. Murine lymphocyte proliferation impaired by substituted neosolaniols and calonectrins — Fusarium metabolites associated with trichothecene biosynthesis. Toxicon 29, 1107 – 1113. Bossy-Wetzel, E., Newmeyer, D.D., Green, D.R., 1998. Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. EMBO J. 17, 37 –49. Carter, C.J., Cannon, M., 1978. Inhibition of eukaryotic ribosomal function by the sesquiterpenoid antibiotic fusarenon-X. Eur. J. Biochem. 84, 103 –111. Cundliffe, E., Cannon, M., Davies, J., 1974. Mechanism of inhibition of eukaryotic protein synthesis by trichothecene fungal toxins. Proc. Nat. Acad. Sci. USA 71, 30 –34. Douglas, R.S., Tarshis, A.D., Pletcher, C.H., Nowell, P.C., Moore, J.S., 1995. A simplified method for the coordinate examination of apoptosis and surface phenotype of murine lymphocytes. J. Immunol. Methods 188, 219 –228. Foa, P., Maiolo, A.T., Lombardi, L., Toivonen, H., Rytoma, T., Polli, E.E., 1982. Growth pattern of the human promyelocytic leukaemia cell line HL60. Cell Tissue Kinet. 15, 399 – 404. Gamen, S., Anel, A., Perez-Galan, P., Lasierra, P., Johonson, D., Pineiro, A., Naval, J., 2000. Doxorubicin treatment activates a Z-VAD-sensitive caspase, which causes Dcm

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loss, caspase-9 activity, and apoptosis in Jurkat cells. Exp. Cell Res. 258, 223 – 235. Gougeon, M.L., Montagnier, L., 1999. Programmed cell death as a mechanism of CD4 and CD8 T cell deletion in AIDS. Molecular control and effect of highly active anti-retroviral therapy. Ann. N.Y. Acad. Sci. 887, 199 – 212. Ihara, T., Sugamata, M., Sekijima, M., Okumura, H., Yoshino, N., Ueno, Y., 1997. Apoptotic cellular damage in mice after T-2 toxin-induced acute toxicosis. Nat. Toxins 5, 141 – 145. Komatsu, N., Oda, T., Muramatsu, T., 1998. Involvement of both caspase-like protease and serin proteases in apoptotic cell death induced by ricin, modeccin, diphtheria toxin, and pseudomonas toxin. J. Biochem. (Tokyo) 124, 1038 – 1044. Li, J., Shimizu, T., 1997. Course of apoptotic changes in the rat gastric mucosa caused by oral administration of fusarenon-X. J. Vet. Med. Sci. 59, 191 – 199. Li, H., Kolluri, S.K., Gu, J., Dawson, M.I., Cao, X., Hobbs, P.D., Lin, B., Chen, G.-Q., Lu, J.-S., Lin, F., Xie, Z., Fontana, J.A., Reed, J.C., Zhang, X.-K., 2000. Cytochrome c release and apoptosis induced by mitochondrial targeting of nuclear orphan receptor TR3. Science 289, 1159 – 1164. Miura, K., Nakajima, Y., Yamanaka, N., Terao, K., Shibato, T., Ishino, S., 1998. Induction of apoptosis with fusarenonX in mouse thymocytes. Toxicology 127, 195 – 206. Murayama, Y., Terao, K., Inoue-Murayama, M., 2000. Molecular cloning and characterization of cynomolgus monkey Fas. Hum. Immunol. 61, 474 – 485. Ohotsubo, K., Saito, M., 1970. Cytotoxic effects of scirpene compounds, fusarenon-X produced by Fusarium ni6ale, dehydronivalenol and dehydrofusarenon-X, on Hela cells. Jpn. J. Med. Sci. Biol. 23, 217 – 225. Ohotsubo, K., Kaden, P., Mittermayer, C., 1972. Polyribosomal breakdown in mouse fibroblast (L-cells) by fusarenonX, a toxic principle isolated from Fusarium ni6ale. Biochim. Biophys. Acta 287, 520 – 525. Okumura, H., Yoshino, N., Ogasawara, Y., Nakamura, K., Ihara, T., Sugamata, M., Takeda, K., Ueno, Y., 1998. Activation of ICE family proteases (caspases) in T-2 toxininduced apoptosis. Mycotoxins 47, 39 – 44. Pestka, J.J., Yan, D., King, L.E., 1994. Flow cytometric analysis of the effects of in 6itro exposure to vomitoxin (deoxynivalenol) on apoptosis in murine T, B and IgA+ cells. Food Chem. Toxicol. 32, 1125 – 1136. Radford, I.R., 1999. Initiation of ionizing radiation-induced apoptosis: DNA damage-mediated or does ceramide have a role? Int. J. Radiat. Biol. 75, 521 – 528. Saito, M., Enomoto, M., Tatsuno, T., 1969. Radiomimetic biological properties of the new scirpene metabolites of Fusarium ni6ale. Gann 60, 599 – 603. Shifrin, V.I., Anderson, P., 1999. Trichothecene mycotoxin triggers a riobotoxic stress response that activates c-Jun N-terminal kinase and p38 mitogen-activated protein kinase and induces apoptosis. J. Biol. Chem. 274, 13985 – 13992.

112

K. Miura et al. / Toxicology 172 (2002) 103–112

Slee, E.A., Keogh, S.A., Martin, S.J., 2000. Cleavage of BID during cytotoxic drug and UV radiation-induced apoptosis occurs downstream of the point of Bcl-2 action and is catalysed by caspase-3: a potential feed-back loop for amplification of apoptosis-associated mitochondrial cytochrome c release. Cell Death Differ. 7, 556 –565. Slee, E.A., Harte, M.T., Kluck, R.M., Wolf, B.B., Casiano, C.A., Newmeyer, D.D., Wang, H.-G., Reed, J., Nicholson, D.W., Alnemri, E.S., Green, D.R., Martin, S.J., 1999. Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspase-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner. J. Cell Biol. 144, 281 – 292. Sun, X.-M, MacFarlane, M., Zhuang, J., Wolf, B.B., Green, D.R., Cohen, G.M., 1999. Distinct caspase cascades are initiated in receptor-mediated and chemical-induced apoptosis. J. Biol. Chem. 274, 5053 –5060. Tandon, H.D., Krogh, P., Ueno, Y., 1990. Trichothecenes. Environmental Health Criteria 105. Selected Mycotoxins: Ochratoxins, Trichothecenes, Ergot. United Nations Environmental Programme, The International Labour Organization and the World Health Organization, Geneva, pp. 71 – 164. Ueno, Y., Ueno, I., Iitoi, Y., Tsynoda, H., Enomoto, M., Ohotsubo, K., 1971. Toxicological approaches in the metabolies of Fusaria III. Acute toxiciy of fusarenon-X. Jpn. J. Exp. Med. 41, 521 –539. Ueno, Y., Nakajima, M., Sakai, K., Ishii, K., Sato, N., Shimada, N., 1973. Comparative toxicology of trichothec

mycotoxins: inhibition of protein synthesis in animal cells. J. Biochem. 74, 285 – 296. Ueno, Y., Umenori, K., Niimi, E., Tanuma, S., Nagata, S., Sugamata, M., Ihara, T., Sekijima, M., Kawai, K., Ueno, I., Tashiro, F., 1995. Induction of apoptosis by T-2 toxin and other natural toxins in HL-60 human promyelotic leukemia cells. Nat. Toxins 3, 129 – 137. Vincenz, C., Dixit, V.M., 1997. Fas-associated death domain protein interleukin-1 beta-converting enzyme 2 (FLICE2), an ICE/Ced-3 homologue, is proximally involved in CD95and p55-mediated death signaling. J. Biol. Chem. 272, 6578 – 6583. Yang, G.H., Jarvis, B.B., Chung, Y.J., Pestka, J.J., 2000. Apoptosis induction by the satratoxins and other trichothecene mycotoxins: relationship to ERK, p38 MAPK, and SAPK/JNK activation. Toxicol. Appl. Pharmacol. 164, 149 – 160. Yang, J., Liu, X., Bhalla, K.B., Kim, C.N., Ibrado, A.M., Cai, J., Peng, T.-I., Jones, D.P., Wang, X., 1997. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275, 1129 – 1132. Zhuang, J., Dinsdale, D., Cohen, G.M., 1998. Apoptosis, in human monocytic THP.1 cells, results in the release of cytochrome c from mitochondria prior to their ultracondensation, formation of outer membrane discontinuities and reduction in inner membrane potential. Cell Death Differ. 5, 953 – 962.