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TOXICOLOGY
B-Related Survival Pathway Down-Regulation
Institut National de la Sante et de la Recherche Medicale U620, Détoxication et Réparation Tissulaire, Université de Rennes 1, Rennes, France (A.L., C.M., O.F., L.V.); Institut National de la Sante et de la Recherche Medicale U517, Mort Cellulaire et Cancer, Facultés de Médecine et Pharmacie, Dijon, France (D.M., O.M.); and Laboratoire d'Hématologie-Immunologie, Centre Hospitalier Universitaire Pontchaillou, Rennes, France (O.F.)
Received July 19, 2005; accepted September 14, 2005.
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Abstract |
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B (NF-B); like As2O3, the selective NF-B inhibitor (E)-3-[(4-methylphenyl)-sulfonyl]-2-propenenitrile (Bay 11-7082) strongly reduced survival of differentiating monocytes. The role of NF-B in arsenic toxicity was also studied in promonocytic U937 cells during phorbol 12-myristate 13-acetate-induced macrophagic differentiation. In these cells, As2O3 first reduced DNA binding of p65 NF-B and subsequently induced apoptosis. In addition, overexpression of the p65 NF-B subunit, following stable infection with a p65 retroviral expressing vector, increased survival of As2O3-treated U937 cells. As2O3 specifically decreased protein levels of X-linked inhibitor of apoptosis protein and FLICE-inhibitory protein, two NF-B-regulated genes in both U937 cells and blood monocytes during their differentiations. Finally, As2O3 was found to inhibit macrophagic differentiation of monocytic cells when used at cytotoxic concentrations; however, overexpression of the p65 NF-B subunit in U937 cells reduced its effects toward differentiation. In contrast to monocytes, well differentiated macrophages were resistant to low concentrations of As2O3. Altogether, our study demonstrates that clinically relevant concentrations of As2O3 induced marked apoptosis of monocytic cells during in vitro macrophagic differentiation likely through inhibition of NF-B-related survival pathways.
). Preliminary clinical data suggest that As2O3 has also a potential effectiveness in patients with other malignant hemopathies, including relapsed or refractory multiple myeloma (Munshi et al., 2002). Hematologic remissions are generally reported using As2O3 at the dose of 0.15 mg/kg per day for variable durations. Following i.v. infusions, blood arsenic levels reach to 5 to 7 µM and then fall approximately to 1 and 0.2 µM at 12 and 24 h after As2O3 administration, respectively (Shen et al., 1997). In vitro, low concentrations of As2O3 are markedly toxic toward acute promyelocytic leukemia and multiple myeloma cells and to other hematological tumoral cells such as non-Hodgkin's lymphoma and chronic lymphocytic leukemia cells (Zhang et al., 1998). Cellular toxicity of As2O3 (0.5–2 µM) can first result from oxidative stress-induced mitochondrial damages and subsequent caspase-dependent apoptosis of leukemia cells (Jing et al., 1999; Zhou et al., 2003). On the other hand, arsenic can also induce apoptosis through inhibition of the NF-B pathway (Mahieux et al., 2001; Mathas et al., 2003; Nasr et al., 2003), which controls expression of various survival proteins, including the caspase inhibitors X-linked inhibitor of apoptosis protein (XIAP) and FLICE-inhibitory protein (FLIP) (Micheau et al., 2001; Zhang et al., 2003). In leukemia cells, arsenic is thought to decrease NF-B activity by directly inhibiting IB
kinase (Mathas et al., 2003), thus preventing NF-B release and translocation to the nucleus.
Besides leukemia cells, As2O3 may also be toxic toward normal hematological cells. Indeed, this metalloid was reported to induce neutropenia in 8% and up to 80% of patients suffering from acute promyelocytic leukemia and multiple myeloma, respectively (Soignet et al., 2001; Munshi et al., 2002). In the case of multiple myeloma, severe cytopenia in As2O3-treated patients is likely related to myelosuppression due to extensive prior therapy; nonetheless, it appears that arsenic can be directly toxic, at least in vitro, to blood-circulating cells. Notably, it was recently reported that low concentrations of As2O3, in the range of clinically effective concentrations (1–5 µM), induce partial apoptosis of T lymphocytes by increasing oxidative stress and caspase activation (Gupta et al., 2003). In addition, sodium arsenite, another trivalent inorganic arsenic salt, was shown to reduce proliferation of normal T lymphocytes at low micromolar concentrations by delaying production and secretion of interleukin-2 (Galicia et al., 2003). Experimental studies have demonstrated that arsenite also markedly impairs functional integrity of monocytes/macrophages. In vivo, it alters macrophage functions such as adhesion or phagocytic activity (Sengupta and Bishayi, 2002) and reduces murine responses against experimental bacterial infection (Bishayi and Sengupta, 2003). In vitro, low concentrations of arsenite affect differentiation of human blood monocytes into mature macrophages, in part by reducing cell viability (Sakurai et al., 2005); molecular pathways mediating arsenic-induced monocytic cell death remain, however, to be determined. In this context, the present study was designed to analyze the effects of As2O3 on survival of human monocytic cells, which constitutes a key biological process of macrophagic differentiation (Kiener et al., 1997; Perlman et al., 1999). Indeed, whereas human monocytes rapidly undergo Fas-mediated apoptosis in vitro, growth factor-induced macrophagic differentiation is associated with increased expression of NF-B-related survival pathways (Perlman et al., 1999; Pennington et al., 2001; Zhang et al., 2003), which could be compromised by As2O3 as reported above.
We demonstrate in this work that clinically relevant concentrations of As2O3 induced marked apoptosis of human blood monocytes and promonocytic U937 cells during macrophagic differentiation induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) and phorbol 12-myristate 13-acetate (PMA), respectively. Apoptosis of monocytic cells likely resulted from decreased NF-B activity and down-regulation of the NF-B-regulated antiapoptotic proteins FLIP and XIAP.
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Materials and Methods |
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B inhibitor, was from Calbiochem (France Biochem, Meudon, France). GM-CSF (specific activity, 1.2 x 108 UI/mg) was obtained from Shering Plough (Levallois-Perret, France), and macrophage colony-stimulating factor (M-CSF) (specific activity, 1 x 105 UI/mg) was from Promocell (Heildeberg, Germany). Rabbit polyclonal antibodies against bcl-xL, caspase-3, IB, mcl-1, and p38 kinase were purchased from Santa Cruz Biotechnology (Tebu-bio S.A., Le Perray en Yvelynes, France). Rabbit polyclonal anti-FLIP antibody was from Stressgen Biotechnologies (Victoria, BC, Canada), whereas mouse monoclonal anti-bcl-2 and anti-XIAP antibodies were obtained from BD Biosciences Pharmingen (San Diego, CA). Mouse monoclonal antibody against caspase-8 was purchased from Alexis Biochemicals (Paris, France). Fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody against CD14, CD71, CD11b, and CD11c was purchased from Immunotech (Marseille, France).
Cells and Treatments
Peripheral blood mononuclear cells were first isolated from bloody buffy coats of healthy donors through Ficoll gradient centrifugation. Human monocytes were then prepared by a 2-h adhesion step, which routinely obtained >90% of adherent CD14-positive cells as assessed by immunostaining. These monocytic cells were next cultured for 6 days in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 20 UI/ml penicillin, and 20 µg/ml streptomycin in the presence of 800 UI/ml GM-CSF or 50 UI/ml M-CSF to get macrophages as previously reported (Young et al., 1990; van Grevenynghe et al., 2003, 2004). The human promonocytic cell line U937 was grown in RPMI 1640 supplemented with 10% fetal bovine serum and was induced to differentiate into macrophage-like cells in the presence of 100 nM PMA during 4 days (Yan et al., 1997). Blood monocytes and U937 cells were induced to differentiate in the absence or presence of As2O3. In some experiments, blood monocytes were first differentiated with GM-CSF for 6 days and then treated with As2O3.
Measurement of Apoptosis and Necrosis
A5 and SG Staining Assays. To analyze alterations of the plasma membrane structure linked to apoptosis, exposition of phosphatidylserine to the extracellular environment was studied. We determined binding of A5, a calcium-dependent protein with high affinity for phosphatidylserine, using A5 conjugated to the fluorescent label Alexa568. Simultaneously, necrotic cells, which have lost their plasma membrane integrity, were detected with the green fluorescent DNA dye SG. Cells were induced to differentiate in the presence or absence of As2O3. Subsequently, cells were collected, washed, and incubated with dyes as previously described (Lemarie et al., 2004). Apoptotic (A5+/SG-) and necrotic (A5-/SG+ and A5+/SG+) cells were quantified using a fluorescence Olympus BX60 microscope (Olympus, Tokyo, Japan) in comparison with living cells. At least 200 cells were counted for each cell suspension.
Hoechst 33342 Staining Assay. To look for changes in chromatin structure typical of apoptotic cells, condensed and fragmented nuclei were stained with the Hoechst 33342 fluorescent DNA dye as previously described (Lemarie et al., 2004). Cells with apoptotic nuclei, i.e., condensed or fragmented, were quantified as described above.
Western Blot Immunoassays
Cells were induced to differentiate into 100-mm dishes in the absence or presence of As2O3. Cells were then harvested, centrifuged, washed with phosphate-buffered saline, and lysed for 20 min on ice in radioimmunoprecipitation assay buffer supplemented with 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 0.5 µg/ml aprotinin, 0.5 mM dithiothreitol, and 1 mM orthovanadate. Cells were then centrifuged at 13,000 rpm for 15 min at 4°C. The resulting supernatants were collected and frozen at -80°C or used immediately. Protein concentration was quantified using the Bradford's method (Bradford, 1976). Each sample (30 µg) or 20 µg of nuclear extract prepared as mentioned below was heated for 5 min at 100°C and then analyzed by 12.5% SDS-polyacrylamide gel electrophoresis and electroblotted overnight onto nitrocellulose membranes (GE Healthcare, Little Chalfont, Buckinghamshire, UK). After blocking, membranes were hybridized with primary antibody overnight at 4°C and washed and incubated with appropriate horseradish peroxidase-conjugated secondary antibody. Immunolabeled proteins were visualized by chemiluminescence.
Caspase Activity Assay
Caspase activity was assessed as previously described (Huc et al., 2004; Lemarie et al., 2004). Crude cell lysate (50 µg) was incubated with 80 µM DEVD-AMC or IETD-AMC, two caspase substrates known to be essentially cleaved by caspase-3 or caspase-8, respectively, for 2 h at 37°C. Caspase-mediated cleavage of substrate-AMC was measured by spectrofluorometry (SpectraMax Gemini; Molecular Devices, Sunnyvale, CA) at the excitation/emission wavelength pair of 380:440 nm. Caspase activities were expressed as the ratio of relative activity of treated cells to that of untreated cells.
Measurement of NF-B DNA Binding
Nuclear proteins were prepared using the Nuclear Extract Kit from Active Motif (Rixensart, Belgium). DNA binding of p65 NF-B was analyzed using the enzyme-linked immunosorbent assay-based TransAM NF-B kit (Active Motif) as previously described (Lemarie et al., 2004). In brief, nuclear cell extracts (10 µg) were incubated for 1 h in a 96-well plate to which oligonucleotide containing an NF-B consensus binding site had been immobilized. After washing, the plate was incubated for 1 h with the rabbit anti-NF-B p65 antibody (1:1000), which specifically detects an epitope accessible only when NF-B p65 is activated and bound to its cognate oligonucleotide. The plate was then washed and incubated with horseradish peroxidase-conjugated secondary antibody (1:1000) for 1 h at room temperature. After washing, colorimetric readout was quantified by spectrophotometry at 450 nm. To monitor the specificity of this assay, wild-type and mutated consensus oligonucleotides were used as competitors for NF-B binding.
Stable Expression of p65 NF-B in U937 Cells
The retroviral vector pMSCV-Puro-p65 was obtained by subcloning the HindIII/HpaI fragment from pEGFP-p65 [kindly provided by Dr. J. Schmid, Center for Biomolecular Medicine and Pharmacology, Medical University Vienna, Austria (Schmid et al., 2000)] into a modified pMSCV-Puro (Clontech; Ozyme, Saint Quentin Yvelines, France) containing HindIII and HpaI in its multiple cloning site. Retroviral production and cell transduction were carried out as previously described (Micheau et al., 2001).
Total RNA Isolation and RT-PCR Assay
Total RNAs were extracted from monocytes using the TRIzol method (Invitrogen, Carlsbad, CA), and RT-PCR analysis was then performed (Laupeze et al., 2002). The primers used for bcl-2, bcl-xL, XIAP, mcl-1, and the long-splice variant of FLIP (FLIPL) have been previously described (Perlman et al., 1999; Cui et al., 2000; Yamaguchi et al., 2002). Glyceraldehyde-3-phosphate dehydrogenase detection was performed as a loading control. PCR products were separated on 1% agarose gel and stained with ethidium bromide.
Flow Cytometric Immunolabeling Assays
After treatment, floating and adherent cells were removed by a 15-min incubation at 37°C in phosphate-buffered saline supplemented with 100 µM ethylenediaminetetraacetic acid, collected, and centrifuged. Then phenotypic analysis of monocytic cells was performed using flow cytometric direct immunofluorescence assays (Laupeze et al., 2002). Fluorescence related to immunolabeling was measured using a FACScalibur flow cytometer (BD Biosciences, San Jose, CA). Each measurement was conducted on 8000 events and analyzed on Cell Quest software (BD Biosciences).
Endocytosis and Phagocytosis Assays
Cells were incubated at 37°C with 1 mg/ml FITC-dextran (Sigma) for 60 min or with 15 µl of fluorescent latex microspheres (Polysciences, Warrington, PA) for 30 min for endocytosis or phagocytosis assays, respectively. Cellular uptake of FITC-dextran and phagocytosis of latex microspheres were then monitored by flow cytometry at 525 nm. Negative controls were performed in parallel by incubating cells with FITC-dextran or latex beads at 4°C instead of 37°C. Each measurement was conducted on 5000 events and analyzed on Cell Quest software.
Statistical Analysis
The results are presented as means ± S.E.M. Significant differences were evaluated with the multirange Dunnett's t test for experiments in which multiple comparisons were studied. Other differences were evaluated with the Student's t test. Criterion of significance of the difference between means was p < 0.05.
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). In these experimental conditions, human monocytes were particularly susceptible to Fas/FasL interaction and subsequent caspase-8-dependent apoptosis (Kiener et al., 1997). Growth factors increased monocyte survival and promoted macrophagic differentiation. Indeed, in the presence of GM-CSF or M-CSF for 6 days, monocytes survived and developed into adherent macrophagic cells displaying a "fried-egg"-like morphology (i.e., large round cells with distinct nuclei) or a spindle-shaped/stellate morphology, respectively (Fig. 1A) (Young et al., 1990). Addition of 1 µMAs2O3 during monocyte differentiation dramatically reduced the number of adherent macrophagic cells and led to an important increase of very small floating cells in culture medium. Using two specific markers of necrosis (SG) and apoptosis (A5), we evaluated global cytotoxicity of As2O3 toward differentiating monocytes. As shown on Fig. 1B, As2O3 induced marked dose-dependent apoptosis (A5+/SG-) of monocytes during their differentiation with GM-CSF for 6 days; 1 µM As2O3 also significantly altered monocyte survival treated with M-CSF. In contrast, as recently reported (Sakurai et al., 2004), neither arsenite (0.5 µM) nor cadmium (0.5–2 µM) significantly altered monocyte viability during differentiation with GM-CSF (data not shown).
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B DNA Binding. As2O3-induced apoptosis appeared to be time-dependent and detectable after 2 days of treatment with GM-CSF (Fig. 2A). Apoptosis was confirmed by chromatin and DNA condensation, as assessed by Hoechst 33342 staining, after a 3-day treatment (Fig. 2B). Accordingly, at this time point, Western blot analysis showed decreased levels of both procaspase-8 and procaspase-3 and apparition of the caspase-3 active cleaved fragment p17 (Fig. 2C). Involvement of caspases was confirmed by measurements of caspase-8 and -3 activities, using IETD-AMC and DEVD-AMC as specific substrates, respectively. Ratios of relative caspase-8 and -3 activities in cells treated for 6 days with 1 µM As2O3 compared with untreated cells were 3.64 ± 0.72 and 3.40 ± 1.25, respectively. Unfortunately, the use of caspase inhibitor to confirm involvement of these cysteine proteases in As2O3-induced apoptosis could not be addressed in our cell system. Indeed, as recently reported in M-CSF-treated monocytes (Sordet et al., 2002), we found that 30 µM z-VAD-fmk shifted differentiation of GM-CSF-treated monocytes to cell death: 9.03 ± 1.8 and 40.4 ± 6% of A5+/SG- cells were measured in untreated and z-VAD-fmk-treated monocytes after 2 days, respectively.
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Different studies report that survival of monocytic cells during growth factor-induced macrophagic differentiation requires NF-B activity (Pennington et al., 2001; Zhang et al., 2003). We thus determined whether As2O3 could alter DNA binding activity of the transcriptionally active p65 NF-B subunit, which is increased during macrophage differentiation (Conti et al., 1997; Ammon et al., 2000). Figure 3A demonstrates that 1 µM As2O3 significantly reduced DNA binding of p65 subunit to B consensus sites by 20 and 50% in GM-CSF-treated monocytes after 3 and 6 days, respectively. In addition, like arsenic, the specific NF-B inhibitor Bay 11-7082 significantly reduced p65 NF-B DNA binding (Fig. 3B), prevented cell adhesion (data not shown), and induced potent apoptosis in GM-CSF-treated monocytes after a 3-day treatment (Fig. 3C). Besides NF-B, we also examined potential roles of p38 kinase and c-Jun N-terminal kinase, two mitogen-activated protein kinases frequently involved in apoptosis induced by inorganic arsenic. Our results demonstrated that neither the p38 kinase inhibitor SB203580 (10 µM) nor the c-Jun N-terminal kinase inhibitor D-JNKI1 (1 µM) could prevent apoptosis of GM-CSF-treated monocytes exposed to As2O3 for 3 days (data not shown).
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B Signals. To further study the role of NF-B in arsenic toxicity toward monocytic cells, we used the promonocytic U937 cell line. Indeed, up-regulation of NF-B pathway confers survival during macrophagic differentiation of U937 cells with PMA (Pennington et al., 2001). As previously described, PMA-induced U937 cell differentiation was associated with rapid cell adhesion and marked expression of the CD11c 
2 integrin after 4 days (data not shown). As2O3 was not toxic toward differentiating U937 cells at 1 µM; in contrast, 4 µMAs2O3 reduced cell adhesion and induced marked apoptosis of differentiating U937 cells (Fig. 4A). Figure 4B indicates that apoptosis was time-dependent and not detectable after 24-h treatment with As2O3.
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B subunit during U937 cell differentiation (Fig. 4C); this effect was associated with a decrease of IB protein levels (Fig. 4D). Addition of As2O3 during PMA-induced differentiation reduced p65 NF-B DNA binding activity in a time-dependent manner (Fig. 4C). This effect was significant as early as 24 h and was associated with a decrease of nuclear levels of p65 NF-B (Fig. 4C, insert) and an inhibition of IB degradation (Fig. 4D). It thus appears that decrease of NF-B DNA binding preceded apoptosis and was not a consequence of cell death. To determine whether NF-B inhibition was a causal event mediating As2O3-induced apoptosis, a p65 NF-B subunit expressing retroviral vector was prepared to stably express p65 in U937 cells; human primary monocytes are difficult to transfect and could not be used in this study. As shown in Fig. 4E, both p65 DNA binding activity and nuclear levels were significantly higher in p65-transduced U937 cells compared with control empty vector, treated or not with PMA. Interestingly, As2O3 toxicity was significantly reduced in p65-overexpressing U937 cells during differentiation with PMA for 48 h (Fig. 4E): percentages of A5+/SG- cells were 28.6 ± 3.7 and 13.6 ± 1.3 in control and p65-overexpressing U937 cells, respectively.
As2O3 and Bay 11-7082 Inhibited FLIP and XIAP Expression during Differentiation. NF-B controls expression of various antiapoptotic proteins, notably FLIP and XIAP (Lin et al., 2001; Micheau et al., 2001; Zhang et al., 2003), two endogenous caspase inhibitors known to increase survival during macrophagic differentiation (Perlman et al., 1999; Lin et al., 2001; Zhang et al., 2003). We analyzed cellular expression of these proteins in response to As2O3 treatment. As shown in Fig. 5A, As2O3 prevented up-regulation of both FLIPL and XIAP in U937 cells during differentiation with PMA without altering mcl-1 expression. Similarly, As2O3 did not significantly impair protein (Fig. 5B) or mRNA (Fig. 5C) levels of the bcl-2, bcl-xL, or mcl-1 antiapoptotic factors in human blood monocytes during differentiation with GM-CSF. In contrast, it markedly inhibited both mRNA and protein levels of FLIPL and XIAP in these cells; Bay 11-7082 also markedly reduced mRNAs levels of FLIPL and XIAP but not those of bcl-2 and bcl-xL in differentiating monocytes (Fig. 5C).
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). At 0.125 µM, a dose that did not alter monocyte survival, no effect was observed on CD71 expression. In addition, 1 µM As203 inhibited expressions of the integrins CD11b and CD11c, two other macrophagic markers (Pennington et al., 2001; van Grevenynghe et al., 2003) (Fig. 6B); it also reduced levels of both endocytosis and phagocytosis, two major functions of macrophages (Fig. 6C). As observed with blood monocytes, cytotoxic concentrations of metalloid altered expression of CD11c in U937 cells during differentiation with PMA; Fig. 6D demonstrates that 4 µM As2O3 prevented up-regulation of CD11c expression, whereas 1 µM As2O3 had no effect. Interestingly, Fig. 6E shows that overexpression of the p65 NF-B subunit in U937 cells not only reduced As2O3-induced apoptosis but also prevented inhibition of CD11c expression.
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Mature Macrophages Are Resistant to As2O3-Induced Apoptosis. Once differentiated, mature macrophages display increased NF-B activity and consequently potently resist to apoptosis (Pagliari et al., 2000). Figure 7, A and B, demonstrates that 1 µM As2O3 neither altered viability of human primary macrophages nor decreased levels of FLIPL or XIAP proteins, respectively; however, a 4-fold higher concentration of As2O3 induced macrophage apoptosis and reduced protein levels of these caspase inhibitors (Fig. 7, A and C).
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Our results demonstrate that As2O3 induced time-dependent apoptosis of human blood monocytes and promonocytic U937 cells during differentiation with GM-CSF and PMA, respectively. Kinetics of death were slow and similar to those reported for human promyelocytic NB4 cells or human lymphoma cells treated with low concentrations of As2O3 (Chen et al., 1997; Mathas et al., 2003). Apoptosis of GM-CSF-treated monocytes was characterized by phosphatidylserine externalization, chromatin condensation, DNA fragmentation, and caspase activation. Involvement of caspases in arsenic-treated monocytes was supported by 1) decreased protein levels of procaspase-8 and procaspase-3 and an increased protein level of the active caspase-3 fragment p17, and 2) increased activity of both caspase-8 and caspase-3. Caspase-8 is an initiator caspase mainly activated within the death-inducing signaling complex by death receptors of the tumor necrosis factor family. It can either directly activate the effector caspase-3 in the cytoplasm and trigger apoptosis or induce disruption of the outer mitochondrial membrane allowing cytochrome c release and activation of effector caspases, including caspase-3, via the apoptosome. Prevention of arsenic toxicity by caspase inhibition could not be tested; indeed, the pan-caspase inhibitor z-VAD-fmk was found, by itself, to shift GM-CSF-induced differentiation to cell death. It thus appears that the level of caspase activity is, in fact, a critical factor since its down-regulation or its excessive up-regulation, like in As2O3-treated monocytes, results in failure of macrophagic differentiation and cell death.
Treatment of monocytic cells with As2O3 was associated with marked reduction of DNA binding activity of the transcriptionally active p65 NF-B subunit. Different arguments support the idea that NF-B inhibition can be a causal event mediating arsenic toxicity toward monocytic cells. First, NF-B activity increases survival of monocytic cells during macrophagic differentiation with M-CSF (Zhang et al., 2003), PMA (Pennington et al., 2001), and likely with GM-CSF since we showed that Bay 11-7082, a selective NF-B inhibitor, induced their apoptosis. Second, our results clearly demonstrate that inhibition of NF-B DNA binding preceded apoptosis and was not a consequence of U937 cell death. Third, increasing DNA binding activity of the p65 NF-B subunit in these cells, following stable infection with a p65-expressing vector, significantly reduced metalloid cytotoxicity during PMA-induced differentiation. The precise mechanism by which As2O3 inhibited NF-B pathway in these cells remains to be further explored. Nevertheless, we show that the metalloid markedly prevented IB degradation in PMA-treated U937 cells; this effect might result from inhibition of IB kinase activity in As2O3-treated U937 cells as previously described in other cell types (Mathas et al., 2003).
NF-B regulates expression of some antiapoptotic proteins, notably FLIP and XIAP, in different cell types (Lin et al., 2001; Micheau et al., 2001; Zhang et al., 2003). Our results suggest that NF-B also controlled expression of these two genes during GM-CSF-induced macrophagic differentiation since Bay 11-7082 specifically reduces their mRNA levels; indeed, this inhibitor had no effect on bcl-2 and bcl-xL gene expressions, which are not regulated by NF-B pathways in mature macrophages (Pagliari et al., 2000). In addition, our results demonstrated that As2O3 selectively reduced mRNA and protein levels of FLIPL and XIAP in differentiating monocytes and U937 cells. Down-regulation of these NF-B-regulated genes did not result from a general toxic effect of As2O3 on transcription, since like Bay 11-7082, it neither alter bcl-2 nor bcl-xL expressions. FLIPL has a strong structure homology to procaspase-8, but it lacks catalytic activity; it directly interacts with procaspase-8 in the death-inducing signaling complex, blocks its cleavage into active fragments, and finally prevents caspase-8-dependent apoptosis (Krueger et al., 2001). Down-regulation of FLIPL expression could thus explain, at least in part, the enhanced caspase-8 activity measured in monocytes exposed to As2O3. XIAP is also a potent suppressor of apoptosis, and its effects are mainly mediated by direct caspase inhibition. It tightly interacts with caspase-9 and -3 but not with caspase-8 (Salvesen and Duckett, 2002). Consequently, arsenic might impair function of both initiator and effector caspases in monocytic cells. In contrast to monocytes, mature macrophages were less sensitive to 1 µM As2O3, which is in agreement with their known resistance to apoptosis. At this concentration, metalloid decreased neither cell viability nor FLIPL protein levels but, unexpectedly, increased those of XIAP. A 4-fold higher concentration (4 µM), however, allowed both FLIPL and XIAP down-regulation and reduced viability of mature macrophages. Altogether, these results suggest that alteration of FLIPL and XIAP expression, likely due to NF-B inhibition, is involved in As2O3 toxicity toward monocytes/macrophages.
Finally, our study shows that As2O3 markedly inhibited macrophagic differentiation of blood monocytes and U937 cells. Different observations strengthen the idea that such an inhibition was mainly related to As2O3-induced apoptosis; first, 0.125 and 1 µM As2O3 modified neither macrophagic marker expressions nor viability in differentiating monocytes and U937 cells, respectively, whereas higher concentrations similarly altered both parameters. Second, blocking of As2O3-induced apoptosis in p65-overexpressing U937 cells allowed CD11c up-regulation during differentiation of U937 cells exposed to As2O3. Differentiation of monocytes into macrophages constitutes a cellular process involved in numerous physiological functions. Indeed, once differentiated, macrophages play a pivotal role in immune defenses by producing cytokines, chemokines, growth factors, or eicosanoids. They also protect against microbial infection and play a role in the tumor cell killing most likely via their ability to present antigens to lymphocytes. In addition, they play an important role in inflammation and lipid metabolism, and bone marrow macrophages are notably involved in erythro-poiesis. Inhibition of macrophagic differentiation by a clinically relevant concentration of As2O3 may therefore lead to deleterious adverse effects in As2O3-treated patients. On the other hand, macrophages participate to the physiopathology of several diseases, such as hemophagocytic syndrome or rheumatoid arthritis (Koch et al., 1994). In these circumstances, As2O3-induced inhibition of macrophagic differentiation may present a clinical interest. In conclusion, our study demonstrated that low clinically achievable concentrations of As2O3 prevented macrophagic differentiation of human monocytic cells by altering NF-B-regulated survival pathways.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: As2O3, arsenic trioxide; NF-B, nuclear factor-B; XIAP, X-linked inhibitor of apoptosis protein; FLIP, FLICE-inhibitory protein; IB, inhibitor of B; GM-CSF, granulocyte-macrophage colony-stimulating factor; PMA, phorbol 12-myristate 13-acetate; SG, Sytox Green; A5, Annexin V; Bay 11-7082, (E)-3-[(4-methylphenyl)-sulfonyl]-2-propenenitrile; M-CSF, macrophage colony-stimulating factor; FITC, fluorescein isothiocyanate; RT-PCR, reverse transcriptase-polymerase chain reaction; FLIPL, long-splice variant of FLIP; DEVD-AMC, Asp-Glu-Val-Asp-amino-4-methylcoumarin; IETD-AMC, Ile-Glu-Thr-Asp-7-amino-4-methyl; z-VAD-fmk, benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole.
Address correspondence to: Laurent Vernhet, Unité INSERM U620, Dé-toxication et Réparation Tissulaire, Faculté des Sciences Pharmaceutiques et Biologiques, Université de Rennes 1, 2 avenue du Pr. Léon Bernard, 35043 Rennes, France. E-mail: laurent.vernhet{at}rennes.inserm.fr
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