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Inhibition of NF-κB by hydroquinone sensitizes human bone marrow progenitor cells to TNF-α-induced apoptosis
Toxicology 187 (2003) 127 /137 www.elsevier.com/locate/toxicol
Inhibition of NF-kB by hydroquinone sensitizes human bone marrow progenitor cells to TNF-a-induced apoptosis Patrick J. Kerzic a, David W. Pyatt a,c, Jia Hua Zheng a, Sherilyn A. Gross a, Anh Le a, Richard D. Irons a,b,d,* a
Molecular Toxicology and Environmental Health Sciences Program, University of Colorado Health Sciences Center, 4200 East 9th Avenue, Box C238, Denver, CO 80262, USA b Department of Pathology, University of Colorado Health Sciences Center, Denver, CO, USA c Department of Biometrics and Preventative Medicine, University of Colorado Health Sciences Center, Denver, CO, USA d Cancer Center, University of Colorado Health Sciences Center, Denver, CO, USA Received 17 September 2002; received in revised form 11 December 2002; accepted 3 February 2003
Abstract Suppression of hematopoiesis is an important mechanism governing blood cell formation. Factors such as tumor necrosis factor alpha (TNF-a) inhibit proliferation and colony-forming activity of bone marrow cells and activate nuclear factor kappa B (NF-kB) in multiple cell types. Activated NF-kB is required for many cells to escape apoptosis, including hematopoietic progenitor cells (HPC). The benzene metabolite hydroquinone (HQ) alters cytokine response and induces cell death in HPC, and inhibits NF-kB activation in T and B cells. Therefore, we studied the potential role of HQ-induced NF-kB inhibition in a hematopoietic cell line (TF-1) and primary HPC in rendering these cells susceptible to TNF-a-induced apoptosis. We demonstrate in both cell types that TNF-a activates NF-kB, and HQ exposure inhibits activation of NF-kB by TNF-a in a dose dependent manner. We further investigated the ability of HQ to potentiate TNF-a-induced apoptosis in these cells, and found that HQ sensitized the cells to the pro-apoptotic effect of TNF-a. These results suggest that NF-kB plays a key role in HPC survival, and that HQ-induced inhibition of NFkB leaves these cells susceptible to cytokine-induced apoptosis. These effects may play a role in the suppression of hematopoiesis seen in some benzene exposed individuals. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Benzene; Hydroquinone; NF-kB; TNF-a; Bone marrow; Apoptosis
1. Introduction
* Corresponding author. Tel.: /1-303-315-7209; fax: /1303-315-7223. E-mail address: [email protected] (R.D. Irons).
Tumor necrosis factor alpha (TNF-a) inhibits the clonogenic activity of hematopoietic progenitor cells (HPC) and can initiate apoptosis in a variety of cell types (Mundle et al., 1999; Zheng et al., 1995; Hehner et al., 1998). In addition to the
0300-483X/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0300-483X(03)00064-7
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role of TNF-a in normal hematopoiesis, it has been implicated in the pathogenesis of certain hematopoietic diseases. For example, increased levels of TNF-a in bone marrow have been found in cases of myelodysplastic syndrome (MDS), which is characterized by rapid cycling of HPC coupled with a high rate of apoptosis (Raza et al., 1997; Tsuchiya et al., 1991). Activation of nuclear factor kappa B (NF-kB) through the TNF-a signaling pathway is a critical signal allowing cells to escape TNF-a-induced apoptosis. NF-kB is a dimer composed of members of the Rel family of proteins which bind to 10 base pair DNA sites following activation by upstream signals (Sen and Baltimore, 1986). NFkB is important for survival of human HPC, which require NF-kB activation for clonogenic response and survival following stimulation with PMA and ionomycin (Pyatt et al., 1999). Inactive NF-kB normally resides in the cytoplasm of the unstimulated cell forming a complex with its inhibitory counterpart I kappa B (IkB). Binding by TNF-a to its receptor initiates a kinase cascade that results in the phosphorylation of IkB at key serine residues leading to its dissociation from NF-kB and ultimate degradation (Traenckner et al., 1995). TNF receptor stimulation results in multiple intracellular signals, one of which is the initiation of an apoptotic signal and another, which normally blocks this cascade, requires activated NFkB. Cells unable to activate NF-kB are sensitive to the pro-apoptotic effects of TNF-a, and cell lines expressing a dominant negative IkB undergo apoptosis when stimulated with TNF-a (Beg and Baltimore, 1996). The benzene metabolite hydroquinone (HQ) is thought to play an important role in bone marrow toxicity associated with benzene and is a potent inhibitor of NF-kB (Pyatt et al., 1998, 2000). HPC exhibit a complex biphasic response to HQ, which enhances cytokine-dependent response at low concentrations (Irons and Stillman, 1996) and produces cytotoxicity at higher concentrations (Moran et al., 1996). In addition, HQ inhibits IL-2 production in stimulated T cells, TNF-a production from stimulated B cells, and IL-1b production from stimulated monocytes (Pyatt et al., 1998, 2000; Carbonnelle et al., 1995). The
effect of HQ on many aspects of HPC function has been documented but is complicated by the relative scarcity of these cells, as they represent approximately 1% of human bone marrow mononuclear cells. For this reason, we and others have employed cell lines such as the human eryhroleukemic TF-1 cells as surrogates in the study of myeloid progenitors. These cells share many characteristics of primary HPC. For example, they are CD34//19/, are responsive to multiple cytokines (Il-3, erythropoietin, and GM-CSF), form colonies in semi-solid support cultures, and can differentiate into multiple mature cell types (Kitamura et al., 1989). Chronic exposure to benzene is associated with bone marrow suppression and a variety of hematologic disorders including MDS and acute myelogenous leukemia in a small proportion of exposed individuals (Linet et al., 1996). Factors such as inter-individual variation in metabolism and detoxication of benzene and its metabolites have been considered as possible explanations for variations in susceptibility to benzene toxicity. However, these variables account for relatively small increases in risk for benzene toxicity (Rothman et al., 1997; Moran et al., 1999). An alternative explanation, that chemicals impart variations in response to hematoregulatory proteins such as TNF-a, is currently under investigation in our laboratory. Here, we tested the hypothesis that TNF-a mediates sensitivity of hematopoietic cells to HQ-induced cytotoxicity via inhibition of NF-kB. Our results demonstrate that NF-kB, normally activated by TNF-a, is inhibited in HQ treated cells, resulting in the induction of apoptosis. These results implicate chemical exposure and cytokine stimulation acting in concert to suppress hematopoiesis, possibly contributing to chemical mediated bone marrow toxicity.
2. Methods 2.1. CD34//19/ cell purification All protocols were approved by the University of Colorado Health Sciences Center Internal Re-
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view Board. Human bone marrow was aspirated from the posterior iliac crest of normal adult volunteers after informed consent. Mononuclear cells were isolated using Histopaque-1077 (Sigma, St. Louis, MO) and CD34//19/ cells were purified using a high magnetic gradient MiniMACS purification system (Miltenyi, Sunnyvale, CA). Depletion of CD19/ cells was performed prior to positive selection for the CD34 antigen using anti-CD19 and -CD34 microbeads, respectively. The purity of isolated cells (/90% CD34//CD19/ for all experiments) was determined using flow cytometric analysis (Coulter Electronics, Hialeah, FL). 2.2. Cell culture and HQ/TNF-a exposure The human erythroleukemia cell line TF-1 was purchased from the American Type Culture Collection (ATCC, Manassas, VA). Purified CD34// 19/ cells or TF-1 cells (1 /5 /105 per ml) were cultured in RPMI 1640 medium (Gibco BRL, Grand Island, NY) supplemented with 10% heatinactivated fetal bovine serum (Gemini Bio-Products, Calabasas, CA), 2 mM L-glutamine, 100 units/ml penicillin (Gibco BRL), and 100 mg/ml streptomycin (Gibco BRL). HQ (Sigma) was dissolved in phosphate-buffered saline (PBS; Gibco BRL) and added to the cells to yield the concentrations indicated. Cells were incubated for 30 min at 37 8C and 5%CO2, and TNF-a (R&D Systems, Minneapolis, MN) was then added to a final concentration of 50 ng/ml, which has been shown to potently activate NF-kB in this population (Pyatt et al., 1999). Cells were incubated for the times indicated at 37 8C and 5%CO2. 2.3. Electrophoretic mobility shift assay (EMSA) Cells were washed in PBS and lysed in lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.02% sodium azide, 0.1% SDS, 100 ug/ml PMSF, 1 ug/ ml aprotinin, 1% nonidet P-40, 0.5% sodium deoxycholate) and protein extracts were frozen at /80 8C until EMSA was conducted. Protein concentrations were determined using a BCA protein kit (Pierce Chemical Co., Rockford, IL). An oligonucleotide (Life Sciences, Grand Island,
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NY) that contains the consensus sequence (GGGGACTTTCCC) recognized by activated NF-kB (Sen and Baltimore, 1986) was annealed to its complementary strand with 5? overhanging ends. The product was labeled with [a-32P]dATP and [a-32P]dGTP using the Klenow subunit of DNA polymerase I (Pharmacia, Piscataway, NJ). The radiolabeled probe was purified using a BioSpin Chromatography Column (Bio-Rad, Hercules, CA) following manufacturer’s instructions. For supershift experiments, extracts were preincubated with 1 ug of antibody to Rel proteins p50, p65, RelB, p52, or c-Rel (Rockland, Gilbertsville, PA) for 1 h on ice. Protein (5 ug) was incubated on ice for 10 min with 1 ug dI /dC, 4 ul binding buffer (20 mM HEPES, pH 7.9, 40 mM KCl, 10% glycerol, 0.05 mM EDTA, 1.6 mM MgCl2), 1 mM DTT, and deionized water for a total volume of 19 ul. One microliter (/50 000 cpm) of 32P-labeled probe was added and the binding reaction was continued at 22 8C for 30 min. For cold competition experiments, reactions were carried out with 100-fold excess unlabeled DNA probe specific for the transcription factors AP-1, NF-kB, or PU.1. Following complex formation 2 ul of EMSA loading buffer (250 mM Tris /HCl (pH 7.5), 0.2% bromophenol blue, 0.2% xylene cyanol, 40% glycerol) was added and the DNA /protein complexes were analyzed by electrophoresis on a 6% polyacrylamide gel. After electrophoresis, gels were dried and exposed at / 80 8C to X-ray film.
2.4. Immunoblotting Proteins from cellular extracts were separated by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (BioRad). The membranes were blocked in TBS with 0.05% Tween 20 and 10% dried milk, and probed with specific horseradish peroxidase-conjugated antibodies (Santa Cruz, Santa Cruz, CA) followed by detection with anti-mouse or anti-rabbit antibodies and exposure using the ECL Plus western blotting detection reagents (Pharmacia).
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2.5. Apoptosis assay Purified CD34//19/ or TF-1 cells were cultured for 16 h under conditions described in the figures, and apoptosis was measured by flow cytometric analysis of deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL, in situ Cell Death Detection Kit, Boerhinger Mannheim). All reported experiments were repeated at least three times.
3. Results 3.1. Identification of TNF-a-induced proteins Human CD34//19/ HPC stimulated with PMA contain NF-kB-specific DNA binding activity comprised of p50, p65, and c-Rel proteins (Pyatt et al., 1999). However, the identity of TNFa-induced NF-kB-specific DNA binding proteins has not been established in this population. Because NF-kB is known to be expressed in B cells, progenitors lacking CD19 were used in these studies. CD34//19/ cells (1 /3/106) were purified and incubated overnight in the presence or absence of 50 ng/ml TNF-a, and cellular extracts were subjected to EMSA using supershift methodologies to determine the identification of Rel proteins present in the DNA-binding complex. TNF-a induced NF-kB-specific DNA binding, and supershift EMSA demonstrated the presence of P50 and P65 (Fig. 1A) as evidenced by the presence of the upper band in the third and fourth lanes. However, no other Rel proteins were activated by TNF-a in primary CD34//19/ cells. The majority of DNA binding is due to p50, while p65 is also present to some degree in the complex. In order to further confirm the identity of the DNA-binding complex, cold competition experiments were carried out with excess unlabeled probe specific for transcription factors known to be present in this population (Fig. 1B). The specificity of NF-kB DNA binding activity is confirmed by the inability of cold AP-1 and PU.1 probes to compete with the radiolabeled NF-kB probe, in contrast with cold NF-kB, which
completely inhibits binding of radiolabeled NFkB. 3.2. HQ inhibits activation of NF-kB by TNF-a in CD34//19/ HPC Inhibition of NF-kB activation by HQ has been observed in T and B cells, resulting in diminished cytokine production (Pyatt et al., 2000, 1998). In order to determine the effect of HQ on TNF-ainduced NF-kB activation in HPC, primary CD34//19/ cells (1 /106 per group) were treated with HQ and TNF-a and harvested after overnight incubation. In primary CD34//19/ cells, HQ exposure resulted in a dose-dependent decrease in NF-kB activation by TNF-a, with 10 uM HQ resulting in complete abrogation of NF-kB DNA binding (Fig. 2). 3.3. HQ sensitizes CD34//19/ and TF-1 cells to TNF-a-induced apoptosis HPC undergo apoptosis when exposed to high concentrations of HQ, and TNF-a is known to induce apoptosis in cells unable to activate NF-kB. In order to determine the consequence of treatment with HQ and TNF-a together on HPC, apoptosis was measured using the TUNEL assay in CD34//19/ cells after 30 min HQ pretreatment and overnight incubation with HQ and TNF-a. The results of three individual experiments are shown. In each experiment, exposure of CD34//19/ cells (2 /105 per group) to TNF-a, 1 uM HQ, or the combination had no effect on apoptosis in CD34//19/ cells. However, treatment with higher doses of HQ together with TNFa led to a greater-than-additive induction of apoptosis over exposure to either agent alone (Fig. 3). The marked variation in the magnitude of the apoptotic response between the three samples demonstrates the wide range of responses within the human population and precludes their analysis as a group. In addition to primary HPC, apoptosis was also confirmed in TF-1 cells using identical culture conditions. In this population TNF-a induced a substantial amount of apoptosis when administered alone, while HQ [10 uM] had a less dramatic effect. However, the combination of
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Fig. 1. Induction of NF-kB-specific DNA binding activity in CD34//19/ cells. (A) CD34//19/ cells (1 /3/106) were exposed to TNF-a (50 ng/ml) overnight, and cell lysates were examined for NF-kB activation. TNF-a induced NF-kB-specific DNA binding, and supershift EMSA demonstrated the presence of P50 and P65 in the NF-kB-specific DNA-binding complex. (B) Cold competition experiments were performed on lysates from TNF-a-activated cells to further confirm specificity of NF-kB DNA binding using excess unlabelled probe. A result representative of three experiments is shown.
the two again led to a greater-than-additive induction of apoptosis (Fig. 4). These results provide further support for the use of TF-1 cells as surrogates for primary HPC in the study of myeloid progenitor cells. 3.4. HQ inhibits NF-kB activation by inhibiting IkB phosphorylation in TF-1 cells Due to the relatively limited number of primary CD34//19/ cells available, the phosphorylation state of IkB-a was examined in TF-1 cells. Unstimulated TF-1 cells display a low level of constitutive activation of NF-kB, and these cells also contained low levels of phosphorylated IkB-a, (p-IkB-a) (Fig. 5A and B). Despite this constitutive activity, both NF-kB activation and phos-
phorylation of IkB-a in response to TNF-a were inhibited by HQ in a dose dependent manner. Complete abrogation of both NF-kB activation and IkB-a phosphorylation was seen at higher levels of HQ in TF-1 cells than in CD34//19/ cells, presumably due to constitutive NF-kB activation in untreated cells. Exposure to HQ had no effect on levels of I kappa kinase subunits alpha and beta (IKK-a, IKK-b) (Fig. 5C and D).
4. Discussion We demonstrate here that TNF-a and HQ cooperate to induce apoptosis in human HPC, and that this effect is likely mediated by the inhibition of NF-kB activation. This is consistent
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Fig. 2. HQ inhibits NF-kB activated by TNF-a in CD34//19/ cells. CD34//19/ cells (1/106 per group) were treated with HQ for 30 min followed by TNF-a, and cells were cultured in the presence of both overnight. NF-kB activation was examined as described in the Section 2. A result representative of three experiments is shown.
with reports demonstrating the requirement for NF-kB activation in preventing TNF-a-induced apoptosis (Beg and Baltimore, 1996; Pyatt et al., 1999; Giri and Aggarwal, 1998; Bellas et al., 1997). These results also implicate a normal regulator of hematopoiesis, TNF-a, in contributing to the hematotoxicity observed in benzene-exposed individuals and suggest a novel potential mechanism for chemical-induced bone marrow toxicity. Multiple observations demonstrate that NF-kB plays a key role in HPC function and is necessary for HPC survival. In human cells, essentially all CD34//19/ HPC exhibit NF-kB DNA binding activity following treatment with PMA, and specific inhibition of NF-kB activation induces both loss of clonogenic response and apoptosis (Pyatt et al., 1999). Further, a number of growth-stimulating cytokines, including IL-3 and GM-CSF, induce NF-kB binding activity in HPC (Pyatt et al., 1999; Tuyt et al., 1996). Knockout studies reveal that mice lacking RelA (p65) die in utero with massive degeneration of the liver, a key hematopoietic organ in the developing mouse, and mice lacking p50 survive gestation but develop multiple defects in immune response (Sha et al., 1995; Gerondakis et al., 1999). Taken together these data suggest a key role for NF-kB in HPC function and survival.
In both human and murine bone marrow, TNF receptor stimulation has been shown to inhibit HPC function, primarily due to activation of the 55 kD TNF receptor, TNFRI (Dybedal et al., 2001). Reports variously attribute inhibition of hematopoiesis either to maintenance of normal progenitor cells in G0/G1 status or by the induction of cell death (Zhang et al., 1995; Selleri et al., 1995). Our data demonstrates cell death in the presence of chemical-induced inhibition of NF-kB activation, suggesting the latter mechanism. Signaling through TNFRI is essential in maintaining normal hematopoiesis, as TNFRI/// mice display a hypercellular marrow and elevated myeloid marrow progenitor and peripheral blood cell counts (Rebel et al., 1999). In humans, TNF-a has been implicated in disease states such as bone marrow failure, MDS, and aplastic anemia. For example, it has been reported that increased levels of TNF-a in the circulation of MDS patients correlates with increased bone marrow cell apoptosis, although its role in mediating these diseases is not well understood (Tsuchiya et al., 1991). The ability of hydroxylated metabolites of benzene to induce apoptosis in HPC has been documented, and this characteristic has made these phenolic compounds the object of much study. However, we demonstrate the induction of
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Fig. 3. HQ sensitizes CD34//19/ cells to TNF-a-induced apoptosis. CD34//19/ cells (2/105 per group) were treated in 1 ml media with the indicated concentrations of HQ for 30 min followed by the addition of 50 ng/ml TNF-a, and cells were cultured with both overnight. Apoptosis was measured using the TUNEL assay. The results of three separate experiments are shown. In control samples no HQ was added.
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Fig. 4. HQ sensitizes TF-1 Cells to TNF-a-induced apoptosis. TF-1 cells were cultured under identical conditions as in Fig. 3, and apoptosis was measured after overnight incubation. Results shown are average 9/S.E.M. for three separate experiments. *, $, %, Represent significant decrease between samples (P 5/0.05).
apoptosis in HPC with HQ and TNF-a at concentrations substantially lower than for HQ alone (10 uM for HQ and TNF-a vs. 100 uM for HQ) (Moran et al., 1996). This combination of low-level exposure and cytokine stimulation may more accurately reflect the in vivo environment of the bone marrow, where HPC are consistently exposed to hematoregulatory proteins influencing their fate. In addition to the induction of apoptosis, benzene metabolites have been demonstrated to alter cytokine response in HPC (Irons and Stillman, 1996). This effect is abrogated in the presence of antioxidants, suggesting that redox cycling may play a role in alterations in hematopoiesis in benzene-exposed individuals (Wiemels and Smith, 1999). However, a pro-oxidant signal from HQ would be expected to activate NF-kB, which is sensitive to oxidative stress. The repeated demon-
stration of inhibition of NF-kB by HQ in T cells, B cells, and HPC is inconsistent with an oxidative mechanism to explain TNF-a-mediated apoptosis in these cells and illustrates the complexity of studying chemical-induced alterations in cell function and hematotoxicity. Chronic exposure to benzene can result in bone marrow suppression in a relatively small number of individuals exposed to high concentrations, indicating that there are important individual differences in susceptibility (Hunter and Hanflig, 1927; Greenburg et al., 1939). In addition, individual susceptibility to bone marrow toxicity is a serious complication of cancer chemotherapy that is difficult to predict. Genetic polymorphisms in metabolism and detoxication have been identified that account for relatively small increases in risk of bone marrow toxicity associated with exposure to some of these agents (Rothman et al., 1997; Moran
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Fig. 5. HQ inhibits TNF-a-induced IkB phosphorylation in TF-1 cells. TF-1 cells (1 /3/106 per group) were treated with HQ for 30 min followed by addition of TNF-a, and cells were cultured in the presence of both for 4 h. Lysates were prepared and analyzed for NF-kB activation, IkB-a phosphorylation, and presence of IKK-a and IKK-b. A result representative of three experiments is shown.
et al., 1999). However, the impact of environmental or genetic influences on the elaboration of regulatory cytokines is largely unknown. Our results suggest that high levels of TNF-a in the bone marrow of individuals exposed to benzene could lead to an excessive apoptotic response, contributing to marrow suppression. A variety of disease states such as rheumatoid arthritis and infection can lead to increases in TNF-a in the blood. Moreover, a polymorphism within the promoter region has been identified that accounts for a wide range of TNF-a production from stimulated blood cells of different individuals, suggesting the possibility that variations in hematoregulatory proteins may play an important role in marrow suppression (Louis et al., 1998). These results indicate that HQ synergizes with TNF-a in producing apoptosis in human bone marrow cells. In addition, these results suggest
that increases in the bone marrow concentration of TNF-a may impact significantly on susceptibility to bone marrow toxicity following exposure to hematotoxic agents. Further characterization of the mechanisms of chemical /cytokine interaction may provide a means to predict individual susceptibility to bone marrow toxicity associated with exposure to these agents.
Acknowledgements The authors thank Ann Louden for assistance with manuscript preparation and Karen Helm for assistance with flow cytometry. This work was supported by NIH grant ES06258 and by the University of Colorado Cancer Center Core NIH grant 2 P30 CA 46934 through the use of its Flow Cytometry and Clinical Investigations Cores.
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Inhibition of NF-kB by hydroquinone sensitizes human bone marrow progenitor cells to TNF-a-induced apoptosis Patrick J. Kerzic a, David W. Pyatt a,c, Jia Hua Zheng a, Sherilyn A. Gross a, Anh Le a, Richard D. Irons a,b,d,* a
Molecular Toxicology and Environmental Health Sciences Program, University of Colorado Health Sciences Center, 4200 East 9th Avenue, Box C238, Denver, CO 80262, USA b Department of Pathology, University of Colorado Health Sciences Center, Denver, CO, USA c Department of Biometrics and Preventative Medicine, University of Colorado Health Sciences Center, Denver, CO, USA d Cancer Center, University of Colorado Health Sciences Center, Denver, CO, USA Received 17 September 2002; received in revised form 11 December 2002; accepted 3 February 2003
Abstract Suppression of hematopoiesis is an important mechanism governing blood cell formation. Factors such as tumor necrosis factor alpha (TNF-a) inhibit proliferation and colony-forming activity of bone marrow cells and activate nuclear factor kappa B (NF-kB) in multiple cell types. Activated NF-kB is required for many cells to escape apoptosis, including hematopoietic progenitor cells (HPC). The benzene metabolite hydroquinone (HQ) alters cytokine response and induces cell death in HPC, and inhibits NF-kB activation in T and B cells. Therefore, we studied the potential role of HQ-induced NF-kB inhibition in a hematopoietic cell line (TF-1) and primary HPC in rendering these cells susceptible to TNF-a-induced apoptosis. We demonstrate in both cell types that TNF-a activates NF-kB, and HQ exposure inhibits activation of NF-kB by TNF-a in a dose dependent manner. We further investigated the ability of HQ to potentiate TNF-a-induced apoptosis in these cells, and found that HQ sensitized the cells to the pro-apoptotic effect of TNF-a. These results suggest that NF-kB plays a key role in HPC survival, and that HQ-induced inhibition of NFkB leaves these cells susceptible to cytokine-induced apoptosis. These effects may play a role in the suppression of hematopoiesis seen in some benzene exposed individuals. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Benzene; Hydroquinone; NF-kB; TNF-a; Bone marrow; Apoptosis
1. Introduction
* Corresponding author. Tel.: /1-303-315-7209; fax: /1303-315-7223. E-mail address: [email protected] (R.D. Irons).
Tumor necrosis factor alpha (TNF-a) inhibits the clonogenic activity of hematopoietic progenitor cells (HPC) and can initiate apoptosis in a variety of cell types (Mundle et al., 1999; Zheng et al., 1995; Hehner et al., 1998). In addition to the
0300-483X/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0300-483X(03)00064-7
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role of TNF-a in normal hematopoiesis, it has been implicated in the pathogenesis of certain hematopoietic diseases. For example, increased levels of TNF-a in bone marrow have been found in cases of myelodysplastic syndrome (MDS), which is characterized by rapid cycling of HPC coupled with a high rate of apoptosis (Raza et al., 1997; Tsuchiya et al., 1991). Activation of nuclear factor kappa B (NF-kB) through the TNF-a signaling pathway is a critical signal allowing cells to escape TNF-a-induced apoptosis. NF-kB is a dimer composed of members of the Rel family of proteins which bind to 10 base pair DNA sites following activation by upstream signals (Sen and Baltimore, 1986). NFkB is important for survival of human HPC, which require NF-kB activation for clonogenic response and survival following stimulation with PMA and ionomycin (Pyatt et al., 1999). Inactive NF-kB normally resides in the cytoplasm of the unstimulated cell forming a complex with its inhibitory counterpart I kappa B (IkB). Binding by TNF-a to its receptor initiates a kinase cascade that results in the phosphorylation of IkB at key serine residues leading to its dissociation from NF-kB and ultimate degradation (Traenckner et al., 1995). TNF receptor stimulation results in multiple intracellular signals, one of which is the initiation of an apoptotic signal and another, which normally blocks this cascade, requires activated NFkB. Cells unable to activate NF-kB are sensitive to the pro-apoptotic effects of TNF-a, and cell lines expressing a dominant negative IkB undergo apoptosis when stimulated with TNF-a (Beg and Baltimore, 1996). The benzene metabolite hydroquinone (HQ) is thought to play an important role in bone marrow toxicity associated with benzene and is a potent inhibitor of NF-kB (Pyatt et al., 1998, 2000). HPC exhibit a complex biphasic response to HQ, which enhances cytokine-dependent response at low concentrations (Irons and Stillman, 1996) and produces cytotoxicity at higher concentrations (Moran et al., 1996). In addition, HQ inhibits IL-2 production in stimulated T cells, TNF-a production from stimulated B cells, and IL-1b production from stimulated monocytes (Pyatt et al., 1998, 2000; Carbonnelle et al., 1995). The
effect of HQ on many aspects of HPC function has been documented but is complicated by the relative scarcity of these cells, as they represent approximately 1% of human bone marrow mononuclear cells. For this reason, we and others have employed cell lines such as the human eryhroleukemic TF-1 cells as surrogates in the study of myeloid progenitors. These cells share many characteristics of primary HPC. For example, they are CD34//19/, are responsive to multiple cytokines (Il-3, erythropoietin, and GM-CSF), form colonies in semi-solid support cultures, and can differentiate into multiple mature cell types (Kitamura et al., 1989). Chronic exposure to benzene is associated with bone marrow suppression and a variety of hematologic disorders including MDS and acute myelogenous leukemia in a small proportion of exposed individuals (Linet et al., 1996). Factors such as inter-individual variation in metabolism and detoxication of benzene and its metabolites have been considered as possible explanations for variations in susceptibility to benzene toxicity. However, these variables account for relatively small increases in risk for benzene toxicity (Rothman et al., 1997; Moran et al., 1999). An alternative explanation, that chemicals impart variations in response to hematoregulatory proteins such as TNF-a, is currently under investigation in our laboratory. Here, we tested the hypothesis that TNF-a mediates sensitivity of hematopoietic cells to HQ-induced cytotoxicity via inhibition of NF-kB. Our results demonstrate that NF-kB, normally activated by TNF-a, is inhibited in HQ treated cells, resulting in the induction of apoptosis. These results implicate chemical exposure and cytokine stimulation acting in concert to suppress hematopoiesis, possibly contributing to chemical mediated bone marrow toxicity.
2. Methods 2.1. CD34//19/ cell purification All protocols were approved by the University of Colorado Health Sciences Center Internal Re-
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view Board. Human bone marrow was aspirated from the posterior iliac crest of normal adult volunteers after informed consent. Mononuclear cells were isolated using Histopaque-1077 (Sigma, St. Louis, MO) and CD34//19/ cells were purified using a high magnetic gradient MiniMACS purification system (Miltenyi, Sunnyvale, CA). Depletion of CD19/ cells was performed prior to positive selection for the CD34 antigen using anti-CD19 and -CD34 microbeads, respectively. The purity of isolated cells (/90% CD34//CD19/ for all experiments) was determined using flow cytometric analysis (Coulter Electronics, Hialeah, FL). 2.2. Cell culture and HQ/TNF-a exposure The human erythroleukemia cell line TF-1 was purchased from the American Type Culture Collection (ATCC, Manassas, VA). Purified CD34// 19/ cells or TF-1 cells (1 /5 /105 per ml) were cultured in RPMI 1640 medium (Gibco BRL, Grand Island, NY) supplemented with 10% heatinactivated fetal bovine serum (Gemini Bio-Products, Calabasas, CA), 2 mM L-glutamine, 100 units/ml penicillin (Gibco BRL), and 100 mg/ml streptomycin (Gibco BRL). HQ (Sigma) was dissolved in phosphate-buffered saline (PBS; Gibco BRL) and added to the cells to yield the concentrations indicated. Cells were incubated for 30 min at 37 8C and 5%CO2, and TNF-a (R&D Systems, Minneapolis, MN) was then added to a final concentration of 50 ng/ml, which has been shown to potently activate NF-kB in this population (Pyatt et al., 1999). Cells were incubated for the times indicated at 37 8C and 5%CO2. 2.3. Electrophoretic mobility shift assay (EMSA) Cells were washed in PBS and lysed in lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.02% sodium azide, 0.1% SDS, 100 ug/ml PMSF, 1 ug/ ml aprotinin, 1% nonidet P-40, 0.5% sodium deoxycholate) and protein extracts were frozen at /80 8C until EMSA was conducted. Protein concentrations were determined using a BCA protein kit (Pierce Chemical Co., Rockford, IL). An oligonucleotide (Life Sciences, Grand Island,
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NY) that contains the consensus sequence (GGGGACTTTCCC) recognized by activated NF-kB (Sen and Baltimore, 1986) was annealed to its complementary strand with 5? overhanging ends. The product was labeled with [a-32P]dATP and [a-32P]dGTP using the Klenow subunit of DNA polymerase I (Pharmacia, Piscataway, NJ). The radiolabeled probe was purified using a BioSpin Chromatography Column (Bio-Rad, Hercules, CA) following manufacturer’s instructions. For supershift experiments, extracts were preincubated with 1 ug of antibody to Rel proteins p50, p65, RelB, p52, or c-Rel (Rockland, Gilbertsville, PA) for 1 h on ice. Protein (5 ug) was incubated on ice for 10 min with 1 ug dI /dC, 4 ul binding buffer (20 mM HEPES, pH 7.9, 40 mM KCl, 10% glycerol, 0.05 mM EDTA, 1.6 mM MgCl2), 1 mM DTT, and deionized water for a total volume of 19 ul. One microliter (/50 000 cpm) of 32P-labeled probe was added and the binding reaction was continued at 22 8C for 30 min. For cold competition experiments, reactions were carried out with 100-fold excess unlabeled DNA probe specific for the transcription factors AP-1, NF-kB, or PU.1. Following complex formation 2 ul of EMSA loading buffer (250 mM Tris /HCl (pH 7.5), 0.2% bromophenol blue, 0.2% xylene cyanol, 40% glycerol) was added and the DNA /protein complexes were analyzed by electrophoresis on a 6% polyacrylamide gel. After electrophoresis, gels were dried and exposed at / 80 8C to X-ray film.
2.4. Immunoblotting Proteins from cellular extracts were separated by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (BioRad). The membranes were blocked in TBS with 0.05% Tween 20 and 10% dried milk, and probed with specific horseradish peroxidase-conjugated antibodies (Santa Cruz, Santa Cruz, CA) followed by detection with anti-mouse or anti-rabbit antibodies and exposure using the ECL Plus western blotting detection reagents (Pharmacia).
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2.5. Apoptosis assay Purified CD34//19/ or TF-1 cells were cultured for 16 h under conditions described in the figures, and apoptosis was measured by flow cytometric analysis of deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL, in situ Cell Death Detection Kit, Boerhinger Mannheim). All reported experiments were repeated at least three times.
3. Results 3.1. Identification of TNF-a-induced proteins Human CD34//19/ HPC stimulated with PMA contain NF-kB-specific DNA binding activity comprised of p50, p65, and c-Rel proteins (Pyatt et al., 1999). However, the identity of TNFa-induced NF-kB-specific DNA binding proteins has not been established in this population. Because NF-kB is known to be expressed in B cells, progenitors lacking CD19 were used in these studies. CD34//19/ cells (1 /3/106) were purified and incubated overnight in the presence or absence of 50 ng/ml TNF-a, and cellular extracts were subjected to EMSA using supershift methodologies to determine the identification of Rel proteins present in the DNA-binding complex. TNF-a induced NF-kB-specific DNA binding, and supershift EMSA demonstrated the presence of P50 and P65 (Fig. 1A) as evidenced by the presence of the upper band in the third and fourth lanes. However, no other Rel proteins were activated by TNF-a in primary CD34//19/ cells. The majority of DNA binding is due to p50, while p65 is also present to some degree in the complex. In order to further confirm the identity of the DNA-binding complex, cold competition experiments were carried out with excess unlabeled probe specific for transcription factors known to be present in this population (Fig. 1B). The specificity of NF-kB DNA binding activity is confirmed by the inability of cold AP-1 and PU.1 probes to compete with the radiolabeled NF-kB probe, in contrast with cold NF-kB, which
completely inhibits binding of radiolabeled NFkB. 3.2. HQ inhibits activation of NF-kB by TNF-a in CD34//19/ HPC Inhibition of NF-kB activation by HQ has been observed in T and B cells, resulting in diminished cytokine production (Pyatt et al., 2000, 1998). In order to determine the effect of HQ on TNF-ainduced NF-kB activation in HPC, primary CD34//19/ cells (1 /106 per group) were treated with HQ and TNF-a and harvested after overnight incubation. In primary CD34//19/ cells, HQ exposure resulted in a dose-dependent decrease in NF-kB activation by TNF-a, with 10 uM HQ resulting in complete abrogation of NF-kB DNA binding (Fig. 2). 3.3. HQ sensitizes CD34//19/ and TF-1 cells to TNF-a-induced apoptosis HPC undergo apoptosis when exposed to high concentrations of HQ, and TNF-a is known to induce apoptosis in cells unable to activate NF-kB. In order to determine the consequence of treatment with HQ and TNF-a together on HPC, apoptosis was measured using the TUNEL assay in CD34//19/ cells after 30 min HQ pretreatment and overnight incubation with HQ and TNF-a. The results of three individual experiments are shown. In each experiment, exposure of CD34//19/ cells (2 /105 per group) to TNF-a, 1 uM HQ, or the combination had no effect on apoptosis in CD34//19/ cells. However, treatment with higher doses of HQ together with TNFa led to a greater-than-additive induction of apoptosis over exposure to either agent alone (Fig. 3). The marked variation in the magnitude of the apoptotic response between the three samples demonstrates the wide range of responses within the human population and precludes their analysis as a group. In addition to primary HPC, apoptosis was also confirmed in TF-1 cells using identical culture conditions. In this population TNF-a induced a substantial amount of apoptosis when administered alone, while HQ [10 uM] had a less dramatic effect. However, the combination of
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Fig. 1. Induction of NF-kB-specific DNA binding activity in CD34//19/ cells. (A) CD34//19/ cells (1 /3/106) were exposed to TNF-a (50 ng/ml) overnight, and cell lysates were examined for NF-kB activation. TNF-a induced NF-kB-specific DNA binding, and supershift EMSA demonstrated the presence of P50 and P65 in the NF-kB-specific DNA-binding complex. (B) Cold competition experiments were performed on lysates from TNF-a-activated cells to further confirm specificity of NF-kB DNA binding using excess unlabelled probe. A result representative of three experiments is shown.
the two again led to a greater-than-additive induction of apoptosis (Fig. 4). These results provide further support for the use of TF-1 cells as surrogates for primary HPC in the study of myeloid progenitor cells. 3.4. HQ inhibits NF-kB activation by inhibiting IkB phosphorylation in TF-1 cells Due to the relatively limited number of primary CD34//19/ cells available, the phosphorylation state of IkB-a was examined in TF-1 cells. Unstimulated TF-1 cells display a low level of constitutive activation of NF-kB, and these cells also contained low levels of phosphorylated IkB-a, (p-IkB-a) (Fig. 5A and B). Despite this constitutive activity, both NF-kB activation and phos-
phorylation of IkB-a in response to TNF-a were inhibited by HQ in a dose dependent manner. Complete abrogation of both NF-kB activation and IkB-a phosphorylation was seen at higher levels of HQ in TF-1 cells than in CD34//19/ cells, presumably due to constitutive NF-kB activation in untreated cells. Exposure to HQ had no effect on levels of I kappa kinase subunits alpha and beta (IKK-a, IKK-b) (Fig. 5C and D).
4. Discussion We demonstrate here that TNF-a and HQ cooperate to induce apoptosis in human HPC, and that this effect is likely mediated by the inhibition of NF-kB activation. This is consistent
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Fig. 2. HQ inhibits NF-kB activated by TNF-a in CD34//19/ cells. CD34//19/ cells (1/106 per group) were treated with HQ for 30 min followed by TNF-a, and cells were cultured in the presence of both overnight. NF-kB activation was examined as described in the Section 2. A result representative of three experiments is shown.
with reports demonstrating the requirement for NF-kB activation in preventing TNF-a-induced apoptosis (Beg and Baltimore, 1996; Pyatt et al., 1999; Giri and Aggarwal, 1998; Bellas et al., 1997). These results also implicate a normal regulator of hematopoiesis, TNF-a, in contributing to the hematotoxicity observed in benzene-exposed individuals and suggest a novel potential mechanism for chemical-induced bone marrow toxicity. Multiple observations demonstrate that NF-kB plays a key role in HPC function and is necessary for HPC survival. In human cells, essentially all CD34//19/ HPC exhibit NF-kB DNA binding activity following treatment with PMA, and specific inhibition of NF-kB activation induces both loss of clonogenic response and apoptosis (Pyatt et al., 1999). Further, a number of growth-stimulating cytokines, including IL-3 and GM-CSF, induce NF-kB binding activity in HPC (Pyatt et al., 1999; Tuyt et al., 1996). Knockout studies reveal that mice lacking RelA (p65) die in utero with massive degeneration of the liver, a key hematopoietic organ in the developing mouse, and mice lacking p50 survive gestation but develop multiple defects in immune response (Sha et al., 1995; Gerondakis et al., 1999). Taken together these data suggest a key role for NF-kB in HPC function and survival.
In both human and murine bone marrow, TNF receptor stimulation has been shown to inhibit HPC function, primarily due to activation of the 55 kD TNF receptor, TNFRI (Dybedal et al., 2001). Reports variously attribute inhibition of hematopoiesis either to maintenance of normal progenitor cells in G0/G1 status or by the induction of cell death (Zhang et al., 1995; Selleri et al., 1995). Our data demonstrates cell death in the presence of chemical-induced inhibition of NF-kB activation, suggesting the latter mechanism. Signaling through TNFRI is essential in maintaining normal hematopoiesis, as TNFRI/// mice display a hypercellular marrow and elevated myeloid marrow progenitor and peripheral blood cell counts (Rebel et al., 1999). In humans, TNF-a has been implicated in disease states such as bone marrow failure, MDS, and aplastic anemia. For example, it has been reported that increased levels of TNF-a in the circulation of MDS patients correlates with increased bone marrow cell apoptosis, although its role in mediating these diseases is not well understood (Tsuchiya et al., 1991). The ability of hydroxylated metabolites of benzene to induce apoptosis in HPC has been documented, and this characteristic has made these phenolic compounds the object of much study. However, we demonstrate the induction of
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Fig. 3. HQ sensitizes CD34//19/ cells to TNF-a-induced apoptosis. CD34//19/ cells (2/105 per group) were treated in 1 ml media with the indicated concentrations of HQ for 30 min followed by the addition of 50 ng/ml TNF-a, and cells were cultured with both overnight. Apoptosis was measured using the TUNEL assay. The results of three separate experiments are shown. In control samples no HQ was added.
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Fig. 4. HQ sensitizes TF-1 Cells to TNF-a-induced apoptosis. TF-1 cells were cultured under identical conditions as in Fig. 3, and apoptosis was measured after overnight incubation. Results shown are average 9/S.E.M. for three separate experiments. *, $, %, Represent significant decrease between samples (P 5/0.05).
apoptosis in HPC with HQ and TNF-a at concentrations substantially lower than for HQ alone (10 uM for HQ and TNF-a vs. 100 uM for HQ) (Moran et al., 1996). This combination of low-level exposure and cytokine stimulation may more accurately reflect the in vivo environment of the bone marrow, where HPC are consistently exposed to hematoregulatory proteins influencing their fate. In addition to the induction of apoptosis, benzene metabolites have been demonstrated to alter cytokine response in HPC (Irons and Stillman, 1996). This effect is abrogated in the presence of antioxidants, suggesting that redox cycling may play a role in alterations in hematopoiesis in benzene-exposed individuals (Wiemels and Smith, 1999). However, a pro-oxidant signal from HQ would be expected to activate NF-kB, which is sensitive to oxidative stress. The repeated demon-
stration of inhibition of NF-kB by HQ in T cells, B cells, and HPC is inconsistent with an oxidative mechanism to explain TNF-a-mediated apoptosis in these cells and illustrates the complexity of studying chemical-induced alterations in cell function and hematotoxicity. Chronic exposure to benzene can result in bone marrow suppression in a relatively small number of individuals exposed to high concentrations, indicating that there are important individual differences in susceptibility (Hunter and Hanflig, 1927; Greenburg et al., 1939). In addition, individual susceptibility to bone marrow toxicity is a serious complication of cancer chemotherapy that is difficult to predict. Genetic polymorphisms in metabolism and detoxication have been identified that account for relatively small increases in risk of bone marrow toxicity associated with exposure to some of these agents (Rothman et al., 1997; Moran
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Fig. 5. HQ inhibits TNF-a-induced IkB phosphorylation in TF-1 cells. TF-1 cells (1 /3/106 per group) were treated with HQ for 30 min followed by addition of TNF-a, and cells were cultured in the presence of both for 4 h. Lysates were prepared and analyzed for NF-kB activation, IkB-a phosphorylation, and presence of IKK-a and IKK-b. A result representative of three experiments is shown.
et al., 1999). However, the impact of environmental or genetic influences on the elaboration of regulatory cytokines is largely unknown. Our results suggest that high levels of TNF-a in the bone marrow of individuals exposed to benzene could lead to an excessive apoptotic response, contributing to marrow suppression. A variety of disease states such as rheumatoid arthritis and infection can lead to increases in TNF-a in the blood. Moreover, a polymorphism within the promoter region has been identified that accounts for a wide range of TNF-a production from stimulated blood cells of different individuals, suggesting the possibility that variations in hematoregulatory proteins may play an important role in marrow suppression (Louis et al., 1998). These results indicate that HQ synergizes with TNF-a in producing apoptosis in human bone marrow cells. In addition, these results suggest
that increases in the bone marrow concentration of TNF-a may impact significantly on susceptibility to bone marrow toxicity following exposure to hematotoxic agents. Further characterization of the mechanisms of chemical /cytokine interaction may provide a means to predict individual susceptibility to bone marrow toxicity associated with exposure to these agents.
Acknowledgements The authors thank Ann Louden for assistance with manuscript preparation and Karen Helm for assistance with flow cytometry. This work was supported by NIH grant ES06258 and by the University of Colorado Cancer Center Core NIH grant 2 P30 CA 46934 through the use of its Flow Cytometry and Clinical Investigations Cores.
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