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Involvement of tumor necrosis factor (TNF-α) in arsenic trioxide induced apoptotic cell death of murine myeloid leukemia cells
Toxicology Letters 135 (2002) 79 – 87 www.elsevier.com/locate/toxlet
Involvement of tumor necrosis factor (TNF-a) in arsenic trioxide induced apoptotic cell death of murine myeloid leukemia cells N.K. Mak a,*, R.N.S. Wong a, K.N. Leung b, M.C. Fung c b
a Department of Biology, The Hong Kong Baptist Uni6ersity, Hong Kong, PR China Department of Biochemistry, The Chinese Uni6ersity of Hong Kong, Hong Kong, PR China c Department of Biology, The Chinese Uni6ersity of Hong Kong, Hong Kong, PR China
Received 21 February 2002; received in revised form 29 May 2002; accepted 29 May 2002
Abstract Arsenic trioxide (As2O3) has recently been shown to be effective to inhibit the growth and to induce apoptosis in acute promyelocytic leukemia (APL) but not in acute myeloid leukemia (AML) cells. Recently, we have isolated an As2O3 sensitive subclone JCS-16 from the murine myeloid leukemia WEHI 3B (JCS). At the concentrations of 0.3– 3 mM, As2O3 induces a dose-dependent cytotoxicity and growth inhibition on the JCS-16 cells. As2O3 also induces apoptotic cell death, as judged by the presence of apoptotic nuclei, at 6 h after treatment. Morphological differentiation was not observed in As2O3 treated JCS cells. Neutralizing anti-TNF-a antibody was found to reduce the As2O3-mediated apoptotic cell death of JCS-16 cells. Growth inhibitory effect of As2O3 was also reduced after the addition of anti-TNF-a. In addition, reverse transcription polymerase chain reaction (RT-PCR) and reverse northern blot analysis demonstrated that the expression of TNF receptor (TNF-R2), IL-4, and IL-4R was down-regulate at 1 h after As2O3 treatment. The expression of TNF-a and TNF-R1 was not affected. Our results suggest that the autocrine action of TNF-a might play a role in As2O3-induced apoptotic cell death of JCS-16 leukemia cells. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Arsenic trioxide; Myeloid leukemia; TNF-a; TNF-R
1. Introduction Arsenic trioxide (As2O3) has recently been evaluated for the treatment of various malignant disorders. These include the various forms of leukemia such as chronic myelogenous leukemia * Corresponding author. Fax: + 86-852-3411-5995 E-mail address: [email protected] (N.K. Mak).
(CML), acute promyelocytic leukemia (APL) and chronic lymphocytic leukemia (CLL), and nonleukemic solid tumors such as neuroblastoma (Akao et al., 1999), malignant cervical (Zheng et al., 1999) and gastric tumors (Zhang et al., 1999). Clinical studies showed that As2O3 is very effective in the treatment of APL. Recently, As2O3 was approved by FDA for the treatment of APL. The specific toxicity of As2O3 on APL cells is due to
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the growth inhibition and cell cycle arrest, induction of apoptosis, and partial induction of differentiation of APL cells (Kitamura et al., 1997; Lu et al., 1999; Zhang et al., 1998). Arsenite-induced apoptosis is triggered by the generation of reactive oxygen species (ROS) which subsequently initiate the programmed cell death (Chen et al., 1998b). In the past decade, we have been studying the role of cytokines in controlling the growth of leukemia cells. We have demonstrated that cytokines, such as IL-1a, IL-1b, IL-3, IL-4, LIF, and tumor necrosis factor (TNF-a) are important in modulating the growth and differentiation of the murine myeloid leukemia WEHI 3B(JCS) and M1 cells (Chen et al., 1997; Fung et al., 1992; Leung et al., 1994; Mak et al., 1993, 1994). TNF-a has been shown to mediate a wide variety of biological effects on leukemia cells. Our earlier studies showed that a low level of TNF-a was constitutively expressed by the WEHI 3B(JCS) (Mak et al., 1993). Recently, it was reported that TNF-a was produced as an inflammatory cytokine from the inorganic arsenicals treated peritoneal macrophages (Sakurai et al., 1998). To further examine the possible biological functions of the inflammatory cytokine TNF-a, we aim to study the influence of TNF-a on As2O3 mediated cytotoxic cell death.
2. Materials and methods
2.1. Cell line and antibody The murine myeloid leukemia WEHI 3B (subclone JCS-16) cell line was used in this study. The WEHI 3B cell line has previously shown to cause an acute nonlymphocytic leukemia in inbred BALB/c mice (Gamba-Vitalo et al., 1989). The cells were grown in RPMI 1640 supplemented with 10% fetal calf serum (FCS, Gibco), 2 mM glutamine, and antibiotics (50 U/ml penicillin, 50 mg/ml streptomycin, and 10 mg/ml neomycin). The cells were incubated at 37 °C in a humidified 5% CO2 incubator. Neutralizing rabbit anti-TNF-a anti-serum was purchased from Genzyme (USA).
2.2. Quantitation of apoptotic cells The apoptotic nuclei were stained with Hoechst 33 258 as described previously (Chen et al., 1998a). Briefly, the arsenite treated leukemia cells were cytocentrifuged onto microscopic slides and fixed with paraformaldehyde (2% in PBS) for 20 min. The cells were washed and stained with Hoechst 33 258 (20 mg/ml) for 15 min. The cells were then observed under a fluorescence microscope (Zeiss Axioplan) with 330–380 nm excitation. The percentage of apoptotic cells was counted. This quantitative measurement had been shown to be consistent with TdT flow cytometry (He et al., 1996).
2.3. Proliferation assay The growth inhibitory effect of As2O3 on the leukemia cells was determined by 3H-thymidine incorporation assay (Mak et al., 2000). Briefly, 1.5× 104 cells were incubated with 0.3–1 mM of As2O3 in 0.2 ml of RPMI-1640 medium supplemented with 10% serum and antibiotics in 96-well flat bottom tissue culture plates for 48 h. During the last 6 h, the cells were incubated with 0.5 mCi 3 H-methyl-thymidine. Cells were harvested onto a glass fiber filter with a cell harvester. The radioactivity was measured using a Beckman scintillation counter.
2.4. Cytotoxicity assay MTT [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide] reduction assay was used to determine the cytotoxicity of As2O3 on leukemia cells (Chen et al., 1998a). The optical density of dissolved formazan crystal was measured using the iEMS Analyzer (Lab-system, Type 1401) at 570 and 690 nm wavelength (Chen et al., 2000a).
2.5. Re6erse transcription polymerase chain reaction (RT-PCR) Total RNA of the arsenic trioxide induced JCS cells was isolated using the guanidine thiocyanate cesium chloride method. The RT-PCR was performed as described previously (Mak et al., 1997).
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Briefly, the RNA samples were quantified by spectrophotometry. The reverse transcription was set up in a 20 ml reaction, containing 1 mg total RNA, 200 U of Moloney murine leukemia virus (MMLV) reverse transcriptase (GibcoBRL), 1× of M-MLV first strand buffer (GibcoBRL), 0.2 mM of each dNTP (Pharmacia), 10 mM of dithiothreitol (GibcoBRL) and 40 U RNasin (Promega), 0.1 mg of oligo d(T)12 – 18 (Pharmacia). RNA samples were incubated at 65 °C for 5 min and quick chill on ice before added to the reaction mix. The reaction mix was incubated at 37 °C for 1 h. PCR was performed in 50 ml reaction containing 0.2 mM of each dNTP, 1 U of Thermoprimeplus DNA polymerase (Advanced biotechnologies), 1.5 mM MgCl2, 1× reaction buffer, 1 pmole of each of the 5% and 3% gene specific primers, and cDNA equivalent to 0.1 mg total RNA. cDNA samples were boiled for 10 min, quick chill on ice before mix with the reaction mixture. The PCR products were electrophoresed on agarose gel, stained with 0.5 mg/ml ethidium bromide and visualized under UV irradiation. To verify the specificity of the TNF-a primers and the PCR products, we have sequenced the PCR products. The sequences of the gene specific PCR primers are shown below: Gene TNF-a
Sequence 5%-TCC CCA AAG GGA TGA GAA GTT C-3%, 5%-TCA TAC CAG GG TTT GAG CTC AG-3% TNF-a 75kD receptor 5%-ATG CCA TGC (TNF-R2) TCA CCG ATT CCA C-3%, 5%-AAC CCG TCT CCT TCC CAC AAC A-3% TNF-a 55kD receptor 5%-CCG AAG TCT (TNF-R1) ACT CCA TCA TTT GT-3%, 5%-ACG CCA TCC ACC ACA GCA TAC-3’ IL-4 5%-TGA CGC ACA GAG CTA TTG ATG G-3%, 5%-ATG ATG CTC TTT AGG CTT TCC AG-3%
IL-4 receptor
18s rRNA
81
5%-CTG GCA CCT GGA GTG AGT GG-3%, 5%-ACA GCG CAC CAC ACT GAC ACT-3% 5%-CGA ACG TCT GCC CTA TCA ACT T-3%, 5%-CGG GCC TGC TTT GAA CAC TCT-3%
2.6. Re6erse northern blot Equal amounts of corresponding gene fragments were denatured with 0.2 M NaOH for 15 min at room temperature and blotted onto five nylon membranes (Boehringer Mannheim) identically. The DNA was fixed by baking at 120 °C for 30 min. cDNA of the induced JCS cells was labeled with DIG-11-dUTP by M-MLV reverse transcriptase in a 20 ml reaction containing 40 U RNasin, 1 × first strand buffer, 200 U M-MLV reverse transcriptase, 10 mM of dithiothreitol, 0.2 mM of each of d-agcTP, 0.13 mM of dTTP, 20 pmole of each of the gene specific primers, 0.07 mM of DIG-11-dUTP (Boehringer Mannheim), and 1 mg RNA. RNA samples were incubated at 65 °C for 5 min and quick chill on ice before adding to the reaction mix. The reaction was carried out at 37 °C for 1 h. 0.3 ml of 0.5 M EDTA was added to terminate the labeling reaction. The labeled cDNA of the induced JCS cells at different time point was heat denatured and hybridized overnight at 42 °C to the immobilized PCR amplified gene fragment in DIG Easy Hyb (Boehringer Mannheim) hybridization solution. The membrane was washed for 5 min in 2× SSC; 0.1% SDS twice at r.t., then washed for 15 min in 0.5× SSC; 0.1% SDS twice at 68 °C. The hybridized DIG-labeled DNA was detected with the chemiluminescent substrate CSPD (Boehringer Mannheim) according to the manufacturer’s instruction. The hybridization signal was visualized by the LUMI-IMAGER™ (Boehringer Mannheim) and quantified by the LUMIANLYST™ software (Boehringer Mannheim). The sequences of the
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gene specific primers for the reverse transcription are shown below:
3.2. Neutralization of As2O3 induced cytotoxicity by anti-TNF-h antibody
Gene TNF-a
To further investigate the role of TNF-a in As2O3-mediated cytotoxicity, anti-TNF-a neutralizing antibody was used in three different assays, namely MTT cytotoxicity assay, anti-leukemia cell proliferation assay, and also in the assay to determine the formation of apoptotic cells. In the MTT cytotoxicity assay, JCS-16 leukemia cells were incubated with various concentrations of As2O3 and neutralizing anti-TNF-a antibody. At the range of 0.3–3 mM, As2O3 killed the JCS-16 leukemia cells in a dose dependent manner (Fig. 2). Significant reduction of cell death by neutralizing anti-TNF-a antibody was observed at a lower concentration (0.3–1 mM) of As2O3. Similar approach was used to measure the 3H-thymidine (3H-TdR) incorporation in the cell proliferation assay. A dose-dependent reduction of proliferation index was observed in As2O3-treated JCS-16 cells. As2O3 (0.3 mM) reduced the proliferation index to 0.29 whereas JCS-16 cells treated with anti-TNF antibody alone had little, if any, change in proliferative index. Co-incubation of JCS-16 with neutralizing TNF-a antibody and As2O3 (0.3 mM) resulted in an increase in the proliferation index from 0.29 to 0.66 (Fig. 3). To determine
TNF-a 75 kDa receptor TNF-a 55 kDa receptor IL-4 IL-4 receptor 18s rRNA
Sequence 5%-AGT TGG TCC CCC TTC TC-3% 5%-GTG TGG GCT TCC GAG AT-3% 5%-AGG CAC GCC ATC CAC C-3% 5%-AGC GCA CCA CAC TGA CA-3% 5%-CTA CGA GTA ATC CAT TTG C-3% 5%-CGG GCC TGC TTT GAA CAC TCT-3%
3. Results
3.1. Apoptotic inducing effects of As2O3 We first studied the apoptotsis-inducing effects of As2O3 on the murine myelomonocytic leukemia WEHI 3B (JCS) cells. Various subclones of JCS cells were tested for the apoptotic response to As2O3. A sensitive subclone, designed JCS-16, was selected in this study. JCS-16 cells were incubated with various concentrations of As2O3 and the apoptotic nuclei were detected using the nuclear stain Hoechst 33 258. The presence of apoptotic nuclei has been demonstrated to correlate with the generation of apoptotic DNA ladder in agarose gel electrophoresis assay (Chen et al., 1998a). Apoptotic nuclei, as judged from the appearance of condensed chromatin and fragmented nuclei, were seen in As2O3 treated JCS-16 cells. Flow cytometric analysis also reveals the presence of sub-G1 peaks, as a result of reduction in DNA content, after As2O3 treatment (data not shown). At the concentration of 1.5 and 3 mM, As2O3 induces a dose-dependent induction of apoptotic cell death (Fig. 1).
Fig. 1. Induction of apoptosis by As2O3. JCS-16 leukemia cells were incubated with various concentrations of As2O3 in wells of 96-well plates. Apoptotic cell counts were determined at 6 h after incubation. At least six hundred cells were counted for each drug concentration. The result was expressed as mean 9 S.D.
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with As2O3 and neutralizing TNF-a antibody. Fig. 4 shows the reduction of apoptotic cell death by the neutralizing antibody. Apoptotic cell death was not seen in the medium control and antibody treatment groups (Fig. 4A and B). Apoptotic cell death, as judged from the presence of apoptotic nuclei, was seen after the treatment with As2O3 (Fig. 4C). In the presence of neutralizing antibody, there was an about 46% reduction of apoptotic cell formation (Fig. 4D).
3.3. mRNA expression profile Fig. 2. Neutralization of As2O3-mediated cytotoxicity by antiTNF-a antibody. JCS-16 cells were co-incubated with various concentrations of As2O3 and neutralizing anti-TNF-a antiserum (diluted 1:50) in wells of 96-well plates. MTT cytotoxicity assay was performed at 24 h after incubation. The result was expressed as mean 9S.D.
whether the reduction of cytotoxicity and the restoration of proliferation capability was associated with the reduced in apoptotic cell death, microscopic examination was carried out to enumerate the number of apoptotic cells in cultures
Fig. 3. Neutralization of As2O3-mediated inhibition of cell growth. JCS-16 cells were co-incubated with various concentrations of As2O3 and neutralizing anti-TNF-a antiserum (diluted 1:50) in wells of 96-well plates. Cell proliferation was determined by the 3H-methyl-thymidine incorporation method. Proliferation index = CPM of the treatment group/ CPM of the control group.
To further determine if the expression of TNF-a and TNF-R in JCS cells was affected during As2O3 treatment, we initially examined the expression profiles of TNF-a, IL-4 and also their corresponding receptors IL-4R, TNF-R1 and TNF-R2 mRNA using reverse Northern dot blot at various times after As2O3 treatment. The TNF-R1 and R2 has been shown to mediate cell death and cell proliferation, respectively (Yuan, 1997). IL-4 has been shown to act synergistically with TNF-a to induce morphological differentiation in this leukemia cell line (Leung et al., 1994). The more stable housekeeping gene 18S rRNA was used as an internal control (Yamada et al., 1997). Since the signal of TNF-a and TNF-R1 was low in the reverse Northern dot blot analysis, the expressions of TNF-a and TNF-R1 mRNA were further examined using PCR method. Table 1 shows the results of reverse Northern dot blot analysis. Reduction of TNF-R2, IL-4, and IL-4R mRNA was observed at 1 h after treatment of the JCS cells with As2O3. The reduction of expressions of TNF-R2 and IL-4R are within the range of 0.659–0.786 in the first 3 h of As2O3 treatment. The reduction of expression of IL-4 is more apparent at 3–4 h post treatment with As2O3. Fig. 5 shows the expression of TNF-a and TNF-R1 in As2O3 treated JCS cells. Consistent with our previous observation (Mak et al., 1993), TNF-a is constitutively expressed in untreated JCS cells. The expression of TNF-a and TNF-R1 was not abolished after As2O3 treatment.
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Fig. 4. Fluorescence images of Hoechst 33258 stained JCS-16 cells. JCS cells were incubated with As2O3 (3 mM) and neutralizing anti-TNF-a antiserum (1:50) in wells of 96-well plates for 6 h. The cells were stained with Hoechst 33258. (A) Untreated control group, (B) anti-TNF-a antiserum, (C) As2O3, and (D) As2O3 and anti-TNF-a antiserum treatment groups. Arrow indicates the apoptotic cell.
4. Discussion TNF-a is a pleiotropic cytokine that mediates a wide variety of biological responses, including the effects on the growth and differentiation of normal and malignant hematopoietic cells, and also the induction of tumor cell death. Macrophages are the major cellular sources of TNF-a. Recent studies showed that TNF-a was produced as a pro-inflammatory cytokine from the inorganic arsenicals treated peritoneal macrophages (Sakurai et al., 1998). We have previously demonstrated that the JCS leukemia cells constitutively express TNF-a mRNA (Mak et al., 1993). In this study, we show that As2O3 induces apoptotsis in JCS cells. To further discern the role of TNF-a in As2O3-mediated leukemia cell death, we applied neutralizing anti-TNF-a antibody in three different sets of experiment. This antibody has previ-
ously been demonstrated to be specific in neutralizing the activity of TNF-a (Mak et al., 1993). Here we demonstrate that the growth inhibitory effect, cytotoxicity, and also the induction of apoptotic cell death by As2O3 are reduced by the neutralizing antibody. Recent research revealed two distinct pathways leading to apoptotic cell death, namely receptor-mediated (e.g. TNF receptor) and mitochondrial-mediated cell death. Two types of TNF receptors have been identified for the TNF, namely TNF-R1 and TNF-R2. The TNF-R1 gene is constitutively expressed at rather low levels (Seitz et al., 1998). The two receptors are independently regulated and transduce distinct intracellular signals. The intracellular death domain of TNF-R1 serves to recruit the adapter protein FADD and the cysteine proteinase caspase-8 (Schneider and Tschopp, 2000). The precursor pro-caspase-8 has recently been found to
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co-localize with apoptogenic protein cytochrome c in mitochondria (Qin et al., 2000). Upon apoptotic stimulation, cytochrome c and pro-caspase-8 are released into cytoplasm. Recent study also shows that arsenite induces apoptosis by causing the generation of ROS and to cause the release of Table 1 Reverse dot blot analysis of gene expression in arsenic trioxide induced JCS cells
Fig. 5. RT-PCR analysis of TNF-a and TNF-a receptor 55 kDa (TNF-R1) gene expression in arsenic trioxide induced JCS cells. 0.1 mg of total RNA of the JCS cells induced for 0 h (0 h), 1 h (1 h), 2 h (2 h), 3 h (3 h) and 4 h (4 h) with As2O3 (1.5 mM) were reverse transcribed by M – MLV reverse transcriptase by oligo d(T)12 – 18. The gene transcripts were amplified by a pair of 3% and 5% sequence specific primers in 30 cycles of PCR. M: 1 kb DNA size marker.
Equal amounts of the corresponding gene fragments were dot blotted onto five nylon membranes identically. One mg of total RNA from untreated or arsenic trioxide (1.5 mM) treated JCS cells (1–4 h) was reverse transcribed into cDNA and labeled with DIG-11-dUTP by M-MLV reverse transcriptase and hybridized to the gene fragments. After washing, chemiluminescent detection was performed. The hybridization signals were quantified using the LUMIANALYST™. Since the expression of the 18S rRNA was relatively stable in both treated and untreated JCS cells, the hybridization signal was normalized using the 18S rRNA. The signal ratio was obtained by comparing the signal intensity of untreated control group with the corresponding treatment groups.
cytochrome c from damaged mitochondria (Chen et al., 1998b). The release of cytochrome c from damaged mitochondria from apoptotic JCS cells has also been reported (Chen et al., 2000b). In this study, neutralizing anti-TNF antibody was found to reduce the apoptotic cell death of JCS leukemia cells. In addition, the expression of TNF-a and TNF-R1 in JCS cells was not inhibited by As2O3 treatment. By analyzing 16 acute myeloid leukemia (AML) cases, it was also found that stimulation of TNF-R1, but not TNF-R2, is responsible for the apoptosis of the AML blasts (Santini et al., 1999). Taken together, both the TNF activation pathway and the mitochondrialmediated pathway might contribute to the As2O3induced apoptotic cell death in the JCS myeloid leukemia cells (Fig. 6). Despite of the induction of partial differentiation in the NB4 APL cell line (Zhang et al., 1998), significant induction of morphological differentiation was not observed in As2O3 treated JCS cells. We have previously demonstrated that IL-4 acts synergistically with TNF-a in inducing the monocytic differentiation of JCS cells (Leung et al., 1994). In this study, the background expression level of IL-4 in JCS cells was about 4-fold reduced
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Fig. 6. Proposed mechanisms for the involvement of TNF-a in arsenite-induced apoptosis. (A) In the absence of caspase 8 endogenously produced TNF-a is not cytotoxic on the JCS leukemia cells. (B) As2O3 induces the production of ROS which in turn causes the release of cytochrome C from damaged mitochondria and the induction of apoptotic cell death (Chen et al., 1998b). Pro-caspase 8, an apoptotic inducer co-localized with cytochrome C in mitochondria (Qin et al., 2000), is released and made available to TNF-R. (C) Reduction of arsenite-induced apoptotic cell death by neutralizing anti-TNF-a antibody in JCS cells indicates the involvement of TNF-a in As2O3-induced apoptotic cell death (this study).
at 4 h after As2O3 treatment. The expression of IL-4R was also down-regulated. The result supports the other observations that As2O3 is not effective in the induction of differentiation of malignant cells (Zhu et al., 1999). The lack of morphological differentiation might be related to the down-regulation of IL-4 and IL-4R in As2O3treated JCS cells. In conclusion, both oxidative stress and the autocrine action of TNF-a might contribute to As2O3 induced apoptotic cell death of JCS-16 myeloid leukemia cells.
Acknowledgements The authors wish to thank Lau Ka Yee for her excellent technical help and statistical analysis.
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Involvement of tumor necrosis factor (TNF-a) in arsenic trioxide induced apoptotic cell death of murine myeloid leukemia cells N.K. Mak a,*, R.N.S. Wong a, K.N. Leung b, M.C. Fung c b
a Department of Biology, The Hong Kong Baptist Uni6ersity, Hong Kong, PR China Department of Biochemistry, The Chinese Uni6ersity of Hong Kong, Hong Kong, PR China c Department of Biology, The Chinese Uni6ersity of Hong Kong, Hong Kong, PR China
Received 21 February 2002; received in revised form 29 May 2002; accepted 29 May 2002
Abstract Arsenic trioxide (As2O3) has recently been shown to be effective to inhibit the growth and to induce apoptosis in acute promyelocytic leukemia (APL) but not in acute myeloid leukemia (AML) cells. Recently, we have isolated an As2O3 sensitive subclone JCS-16 from the murine myeloid leukemia WEHI 3B (JCS). At the concentrations of 0.3– 3 mM, As2O3 induces a dose-dependent cytotoxicity and growth inhibition on the JCS-16 cells. As2O3 also induces apoptotic cell death, as judged by the presence of apoptotic nuclei, at 6 h after treatment. Morphological differentiation was not observed in As2O3 treated JCS cells. Neutralizing anti-TNF-a antibody was found to reduce the As2O3-mediated apoptotic cell death of JCS-16 cells. Growth inhibitory effect of As2O3 was also reduced after the addition of anti-TNF-a. In addition, reverse transcription polymerase chain reaction (RT-PCR) and reverse northern blot analysis demonstrated that the expression of TNF receptor (TNF-R2), IL-4, and IL-4R was down-regulate at 1 h after As2O3 treatment. The expression of TNF-a and TNF-R1 was not affected. Our results suggest that the autocrine action of TNF-a might play a role in As2O3-induced apoptotic cell death of JCS-16 leukemia cells. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Arsenic trioxide; Myeloid leukemia; TNF-a; TNF-R
1. Introduction Arsenic trioxide (As2O3) has recently been evaluated for the treatment of various malignant disorders. These include the various forms of leukemia such as chronic myelogenous leukemia * Corresponding author. Fax: + 86-852-3411-5995 E-mail address: [email protected] (N.K. Mak).
(CML), acute promyelocytic leukemia (APL) and chronic lymphocytic leukemia (CLL), and nonleukemic solid tumors such as neuroblastoma (Akao et al., 1999), malignant cervical (Zheng et al., 1999) and gastric tumors (Zhang et al., 1999). Clinical studies showed that As2O3 is very effective in the treatment of APL. Recently, As2O3 was approved by FDA for the treatment of APL. The specific toxicity of As2O3 on APL cells is due to
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the growth inhibition and cell cycle arrest, induction of apoptosis, and partial induction of differentiation of APL cells (Kitamura et al., 1997; Lu et al., 1999; Zhang et al., 1998). Arsenite-induced apoptosis is triggered by the generation of reactive oxygen species (ROS) which subsequently initiate the programmed cell death (Chen et al., 1998b). In the past decade, we have been studying the role of cytokines in controlling the growth of leukemia cells. We have demonstrated that cytokines, such as IL-1a, IL-1b, IL-3, IL-4, LIF, and tumor necrosis factor (TNF-a) are important in modulating the growth and differentiation of the murine myeloid leukemia WEHI 3B(JCS) and M1 cells (Chen et al., 1997; Fung et al., 1992; Leung et al., 1994; Mak et al., 1993, 1994). TNF-a has been shown to mediate a wide variety of biological effects on leukemia cells. Our earlier studies showed that a low level of TNF-a was constitutively expressed by the WEHI 3B(JCS) (Mak et al., 1993). Recently, it was reported that TNF-a was produced as an inflammatory cytokine from the inorganic arsenicals treated peritoneal macrophages (Sakurai et al., 1998). To further examine the possible biological functions of the inflammatory cytokine TNF-a, we aim to study the influence of TNF-a on As2O3 mediated cytotoxic cell death.
2. Materials and methods
2.1. Cell line and antibody The murine myeloid leukemia WEHI 3B (subclone JCS-16) cell line was used in this study. The WEHI 3B cell line has previously shown to cause an acute nonlymphocytic leukemia in inbred BALB/c mice (Gamba-Vitalo et al., 1989). The cells were grown in RPMI 1640 supplemented with 10% fetal calf serum (FCS, Gibco), 2 mM glutamine, and antibiotics (50 U/ml penicillin, 50 mg/ml streptomycin, and 10 mg/ml neomycin). The cells were incubated at 37 °C in a humidified 5% CO2 incubator. Neutralizing rabbit anti-TNF-a anti-serum was purchased from Genzyme (USA).
2.2. Quantitation of apoptotic cells The apoptotic nuclei were stained with Hoechst 33 258 as described previously (Chen et al., 1998a). Briefly, the arsenite treated leukemia cells were cytocentrifuged onto microscopic slides and fixed with paraformaldehyde (2% in PBS) for 20 min. The cells were washed and stained with Hoechst 33 258 (20 mg/ml) for 15 min. The cells were then observed under a fluorescence microscope (Zeiss Axioplan) with 330–380 nm excitation. The percentage of apoptotic cells was counted. This quantitative measurement had been shown to be consistent with TdT flow cytometry (He et al., 1996).
2.3. Proliferation assay The growth inhibitory effect of As2O3 on the leukemia cells was determined by 3H-thymidine incorporation assay (Mak et al., 2000). Briefly, 1.5× 104 cells were incubated with 0.3–1 mM of As2O3 in 0.2 ml of RPMI-1640 medium supplemented with 10% serum and antibiotics in 96-well flat bottom tissue culture plates for 48 h. During the last 6 h, the cells were incubated with 0.5 mCi 3 H-methyl-thymidine. Cells were harvested onto a glass fiber filter with a cell harvester. The radioactivity was measured using a Beckman scintillation counter.
2.4. Cytotoxicity assay MTT [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide] reduction assay was used to determine the cytotoxicity of As2O3 on leukemia cells (Chen et al., 1998a). The optical density of dissolved formazan crystal was measured using the iEMS Analyzer (Lab-system, Type 1401) at 570 and 690 nm wavelength (Chen et al., 2000a).
2.5. Re6erse transcription polymerase chain reaction (RT-PCR) Total RNA of the arsenic trioxide induced JCS cells was isolated using the guanidine thiocyanate cesium chloride method. The RT-PCR was performed as described previously (Mak et al., 1997).
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Briefly, the RNA samples were quantified by spectrophotometry. The reverse transcription was set up in a 20 ml reaction, containing 1 mg total RNA, 200 U of Moloney murine leukemia virus (MMLV) reverse transcriptase (GibcoBRL), 1× of M-MLV first strand buffer (GibcoBRL), 0.2 mM of each dNTP (Pharmacia), 10 mM of dithiothreitol (GibcoBRL) and 40 U RNasin (Promega), 0.1 mg of oligo d(T)12 – 18 (Pharmacia). RNA samples were incubated at 65 °C for 5 min and quick chill on ice before added to the reaction mix. The reaction mix was incubated at 37 °C for 1 h. PCR was performed in 50 ml reaction containing 0.2 mM of each dNTP, 1 U of Thermoprimeplus DNA polymerase (Advanced biotechnologies), 1.5 mM MgCl2, 1× reaction buffer, 1 pmole of each of the 5% and 3% gene specific primers, and cDNA equivalent to 0.1 mg total RNA. cDNA samples were boiled for 10 min, quick chill on ice before mix with the reaction mixture. The PCR products were electrophoresed on agarose gel, stained with 0.5 mg/ml ethidium bromide and visualized under UV irradiation. To verify the specificity of the TNF-a primers and the PCR products, we have sequenced the PCR products. The sequences of the gene specific PCR primers are shown below: Gene TNF-a
Sequence 5%-TCC CCA AAG GGA TGA GAA GTT C-3%, 5%-TCA TAC CAG GG TTT GAG CTC AG-3% TNF-a 75kD receptor 5%-ATG CCA TGC (TNF-R2) TCA CCG ATT CCA C-3%, 5%-AAC CCG TCT CCT TCC CAC AAC A-3% TNF-a 55kD receptor 5%-CCG AAG TCT (TNF-R1) ACT CCA TCA TTT GT-3%, 5%-ACG CCA TCC ACC ACA GCA TAC-3’ IL-4 5%-TGA CGC ACA GAG CTA TTG ATG G-3%, 5%-ATG ATG CTC TTT AGG CTT TCC AG-3%
IL-4 receptor
18s rRNA
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5%-CTG GCA CCT GGA GTG AGT GG-3%, 5%-ACA GCG CAC CAC ACT GAC ACT-3% 5%-CGA ACG TCT GCC CTA TCA ACT T-3%, 5%-CGG GCC TGC TTT GAA CAC TCT-3%
2.6. Re6erse northern blot Equal amounts of corresponding gene fragments were denatured with 0.2 M NaOH for 15 min at room temperature and blotted onto five nylon membranes (Boehringer Mannheim) identically. The DNA was fixed by baking at 120 °C for 30 min. cDNA of the induced JCS cells was labeled with DIG-11-dUTP by M-MLV reverse transcriptase in a 20 ml reaction containing 40 U RNasin, 1 × first strand buffer, 200 U M-MLV reverse transcriptase, 10 mM of dithiothreitol, 0.2 mM of each of d-agcTP, 0.13 mM of dTTP, 20 pmole of each of the gene specific primers, 0.07 mM of DIG-11-dUTP (Boehringer Mannheim), and 1 mg RNA. RNA samples were incubated at 65 °C for 5 min and quick chill on ice before adding to the reaction mix. The reaction was carried out at 37 °C for 1 h. 0.3 ml of 0.5 M EDTA was added to terminate the labeling reaction. The labeled cDNA of the induced JCS cells at different time point was heat denatured and hybridized overnight at 42 °C to the immobilized PCR amplified gene fragment in DIG Easy Hyb (Boehringer Mannheim) hybridization solution. The membrane was washed for 5 min in 2× SSC; 0.1% SDS twice at r.t., then washed for 15 min in 0.5× SSC; 0.1% SDS twice at 68 °C. The hybridized DIG-labeled DNA was detected with the chemiluminescent substrate CSPD (Boehringer Mannheim) according to the manufacturer’s instruction. The hybridization signal was visualized by the LUMI-IMAGER™ (Boehringer Mannheim) and quantified by the LUMIANLYST™ software (Boehringer Mannheim). The sequences of the
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gene specific primers for the reverse transcription are shown below:
3.2. Neutralization of As2O3 induced cytotoxicity by anti-TNF-h antibody
Gene TNF-a
To further investigate the role of TNF-a in As2O3-mediated cytotoxicity, anti-TNF-a neutralizing antibody was used in three different assays, namely MTT cytotoxicity assay, anti-leukemia cell proliferation assay, and also in the assay to determine the formation of apoptotic cells. In the MTT cytotoxicity assay, JCS-16 leukemia cells were incubated with various concentrations of As2O3 and neutralizing anti-TNF-a antibody. At the range of 0.3–3 mM, As2O3 killed the JCS-16 leukemia cells in a dose dependent manner (Fig. 2). Significant reduction of cell death by neutralizing anti-TNF-a antibody was observed at a lower concentration (0.3–1 mM) of As2O3. Similar approach was used to measure the 3H-thymidine (3H-TdR) incorporation in the cell proliferation assay. A dose-dependent reduction of proliferation index was observed in As2O3-treated JCS-16 cells. As2O3 (0.3 mM) reduced the proliferation index to 0.29 whereas JCS-16 cells treated with anti-TNF antibody alone had little, if any, change in proliferative index. Co-incubation of JCS-16 with neutralizing TNF-a antibody and As2O3 (0.3 mM) resulted in an increase in the proliferation index from 0.29 to 0.66 (Fig. 3). To determine
TNF-a 75 kDa receptor TNF-a 55 kDa receptor IL-4 IL-4 receptor 18s rRNA
Sequence 5%-AGT TGG TCC CCC TTC TC-3% 5%-GTG TGG GCT TCC GAG AT-3% 5%-AGG CAC GCC ATC CAC C-3% 5%-AGC GCA CCA CAC TGA CA-3% 5%-CTA CGA GTA ATC CAT TTG C-3% 5%-CGG GCC TGC TTT GAA CAC TCT-3%
3. Results
3.1. Apoptotic inducing effects of As2O3 We first studied the apoptotsis-inducing effects of As2O3 on the murine myelomonocytic leukemia WEHI 3B (JCS) cells. Various subclones of JCS cells were tested for the apoptotic response to As2O3. A sensitive subclone, designed JCS-16, was selected in this study. JCS-16 cells were incubated with various concentrations of As2O3 and the apoptotic nuclei were detected using the nuclear stain Hoechst 33 258. The presence of apoptotic nuclei has been demonstrated to correlate with the generation of apoptotic DNA ladder in agarose gel electrophoresis assay (Chen et al., 1998a). Apoptotic nuclei, as judged from the appearance of condensed chromatin and fragmented nuclei, were seen in As2O3 treated JCS-16 cells. Flow cytometric analysis also reveals the presence of sub-G1 peaks, as a result of reduction in DNA content, after As2O3 treatment (data not shown). At the concentration of 1.5 and 3 mM, As2O3 induces a dose-dependent induction of apoptotic cell death (Fig. 1).
Fig. 1. Induction of apoptosis by As2O3. JCS-16 leukemia cells were incubated with various concentrations of As2O3 in wells of 96-well plates. Apoptotic cell counts were determined at 6 h after incubation. At least six hundred cells were counted for each drug concentration. The result was expressed as mean 9 S.D.
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with As2O3 and neutralizing TNF-a antibody. Fig. 4 shows the reduction of apoptotic cell death by the neutralizing antibody. Apoptotic cell death was not seen in the medium control and antibody treatment groups (Fig. 4A and B). Apoptotic cell death, as judged from the presence of apoptotic nuclei, was seen after the treatment with As2O3 (Fig. 4C). In the presence of neutralizing antibody, there was an about 46% reduction of apoptotic cell formation (Fig. 4D).
3.3. mRNA expression profile Fig. 2. Neutralization of As2O3-mediated cytotoxicity by antiTNF-a antibody. JCS-16 cells were co-incubated with various concentrations of As2O3 and neutralizing anti-TNF-a antiserum (diluted 1:50) in wells of 96-well plates. MTT cytotoxicity assay was performed at 24 h after incubation. The result was expressed as mean 9S.D.
whether the reduction of cytotoxicity and the restoration of proliferation capability was associated with the reduced in apoptotic cell death, microscopic examination was carried out to enumerate the number of apoptotic cells in cultures
Fig. 3. Neutralization of As2O3-mediated inhibition of cell growth. JCS-16 cells were co-incubated with various concentrations of As2O3 and neutralizing anti-TNF-a antiserum (diluted 1:50) in wells of 96-well plates. Cell proliferation was determined by the 3H-methyl-thymidine incorporation method. Proliferation index = CPM of the treatment group/ CPM of the control group.
To further determine if the expression of TNF-a and TNF-R in JCS cells was affected during As2O3 treatment, we initially examined the expression profiles of TNF-a, IL-4 and also their corresponding receptors IL-4R, TNF-R1 and TNF-R2 mRNA using reverse Northern dot blot at various times after As2O3 treatment. The TNF-R1 and R2 has been shown to mediate cell death and cell proliferation, respectively (Yuan, 1997). IL-4 has been shown to act synergistically with TNF-a to induce morphological differentiation in this leukemia cell line (Leung et al., 1994). The more stable housekeeping gene 18S rRNA was used as an internal control (Yamada et al., 1997). Since the signal of TNF-a and TNF-R1 was low in the reverse Northern dot blot analysis, the expressions of TNF-a and TNF-R1 mRNA were further examined using PCR method. Table 1 shows the results of reverse Northern dot blot analysis. Reduction of TNF-R2, IL-4, and IL-4R mRNA was observed at 1 h after treatment of the JCS cells with As2O3. The reduction of expressions of TNF-R2 and IL-4R are within the range of 0.659–0.786 in the first 3 h of As2O3 treatment. The reduction of expression of IL-4 is more apparent at 3–4 h post treatment with As2O3. Fig. 5 shows the expression of TNF-a and TNF-R1 in As2O3 treated JCS cells. Consistent with our previous observation (Mak et al., 1993), TNF-a is constitutively expressed in untreated JCS cells. The expression of TNF-a and TNF-R1 was not abolished after As2O3 treatment.
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Fig. 4. Fluorescence images of Hoechst 33258 stained JCS-16 cells. JCS cells were incubated with As2O3 (3 mM) and neutralizing anti-TNF-a antiserum (1:50) in wells of 96-well plates for 6 h. The cells were stained with Hoechst 33258. (A) Untreated control group, (B) anti-TNF-a antiserum, (C) As2O3, and (D) As2O3 and anti-TNF-a antiserum treatment groups. Arrow indicates the apoptotic cell.
4. Discussion TNF-a is a pleiotropic cytokine that mediates a wide variety of biological responses, including the effects on the growth and differentiation of normal and malignant hematopoietic cells, and also the induction of tumor cell death. Macrophages are the major cellular sources of TNF-a. Recent studies showed that TNF-a was produced as a pro-inflammatory cytokine from the inorganic arsenicals treated peritoneal macrophages (Sakurai et al., 1998). We have previously demonstrated that the JCS leukemia cells constitutively express TNF-a mRNA (Mak et al., 1993). In this study, we show that As2O3 induces apoptotsis in JCS cells. To further discern the role of TNF-a in As2O3-mediated leukemia cell death, we applied neutralizing anti-TNF-a antibody in three different sets of experiment. This antibody has previ-
ously been demonstrated to be specific in neutralizing the activity of TNF-a (Mak et al., 1993). Here we demonstrate that the growth inhibitory effect, cytotoxicity, and also the induction of apoptotic cell death by As2O3 are reduced by the neutralizing antibody. Recent research revealed two distinct pathways leading to apoptotic cell death, namely receptor-mediated (e.g. TNF receptor) and mitochondrial-mediated cell death. Two types of TNF receptors have been identified for the TNF, namely TNF-R1 and TNF-R2. The TNF-R1 gene is constitutively expressed at rather low levels (Seitz et al., 1998). The two receptors are independently regulated and transduce distinct intracellular signals. The intracellular death domain of TNF-R1 serves to recruit the adapter protein FADD and the cysteine proteinase caspase-8 (Schneider and Tschopp, 2000). The precursor pro-caspase-8 has recently been found to
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co-localize with apoptogenic protein cytochrome c in mitochondria (Qin et al., 2000). Upon apoptotic stimulation, cytochrome c and pro-caspase-8 are released into cytoplasm. Recent study also shows that arsenite induces apoptosis by causing the generation of ROS and to cause the release of Table 1 Reverse dot blot analysis of gene expression in arsenic trioxide induced JCS cells
Fig. 5. RT-PCR analysis of TNF-a and TNF-a receptor 55 kDa (TNF-R1) gene expression in arsenic trioxide induced JCS cells. 0.1 mg of total RNA of the JCS cells induced for 0 h (0 h), 1 h (1 h), 2 h (2 h), 3 h (3 h) and 4 h (4 h) with As2O3 (1.5 mM) were reverse transcribed by M – MLV reverse transcriptase by oligo d(T)12 – 18. The gene transcripts were amplified by a pair of 3% and 5% sequence specific primers in 30 cycles of PCR. M: 1 kb DNA size marker.
Equal amounts of the corresponding gene fragments were dot blotted onto five nylon membranes identically. One mg of total RNA from untreated or arsenic trioxide (1.5 mM) treated JCS cells (1–4 h) was reverse transcribed into cDNA and labeled with DIG-11-dUTP by M-MLV reverse transcriptase and hybridized to the gene fragments. After washing, chemiluminescent detection was performed. The hybridization signals were quantified using the LUMIANALYST™. Since the expression of the 18S rRNA was relatively stable in both treated and untreated JCS cells, the hybridization signal was normalized using the 18S rRNA. The signal ratio was obtained by comparing the signal intensity of untreated control group with the corresponding treatment groups.
cytochrome c from damaged mitochondria (Chen et al., 1998b). The release of cytochrome c from damaged mitochondria from apoptotic JCS cells has also been reported (Chen et al., 2000b). In this study, neutralizing anti-TNF antibody was found to reduce the apoptotic cell death of JCS leukemia cells. In addition, the expression of TNF-a and TNF-R1 in JCS cells was not inhibited by As2O3 treatment. By analyzing 16 acute myeloid leukemia (AML) cases, it was also found that stimulation of TNF-R1, but not TNF-R2, is responsible for the apoptosis of the AML blasts (Santini et al., 1999). Taken together, both the TNF activation pathway and the mitochondrialmediated pathway might contribute to the As2O3induced apoptotic cell death in the JCS myeloid leukemia cells (Fig. 6). Despite of the induction of partial differentiation in the NB4 APL cell line (Zhang et al., 1998), significant induction of morphological differentiation was not observed in As2O3 treated JCS cells. We have previously demonstrated that IL-4 acts synergistically with TNF-a in inducing the monocytic differentiation of JCS cells (Leung et al., 1994). In this study, the background expression level of IL-4 in JCS cells was about 4-fold reduced
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Fig. 6. Proposed mechanisms for the involvement of TNF-a in arsenite-induced apoptosis. (A) In the absence of caspase 8 endogenously produced TNF-a is not cytotoxic on the JCS leukemia cells. (B) As2O3 induces the production of ROS which in turn causes the release of cytochrome C from damaged mitochondria and the induction of apoptotic cell death (Chen et al., 1998b). Pro-caspase 8, an apoptotic inducer co-localized with cytochrome C in mitochondria (Qin et al., 2000), is released and made available to TNF-R. (C) Reduction of arsenite-induced apoptotic cell death by neutralizing anti-TNF-a antibody in JCS cells indicates the involvement of TNF-a in As2O3-induced apoptotic cell death (this study).
at 4 h after As2O3 treatment. The expression of IL-4R was also down-regulated. The result supports the other observations that As2O3 is not effective in the induction of differentiation of malignant cells (Zhu et al., 1999). The lack of morphological differentiation might be related to the down-regulation of IL-4 and IL-4R in As2O3treated JCS cells. In conclusion, both oxidative stress and the autocrine action of TNF-a might contribute to As2O3 induced apoptotic cell death of JCS-16 myeloid leukemia cells.
Acknowledgements The authors wish to thank Lau Ka Yee for her excellent technical help and statistical analysis.
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