From the Departments of Thoracic/Head and Neck
Medical Oncology and § Thoracic and Cardiovascular Surgery,
The University of Texas M. D. Anderson Cancer Center, Houston,
Texas 77030 and the ¶ Retinoid Program, SRI International,
Menlo Park, California 94025
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ABSTRACT |
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Retinoids, including retinol and retinoic acid
derivatives, inhibit the growth of normal human bronchial epithelial
(HBE) cells. The signaling pathways through which retinoids mediate this effect have not been defined. Normal HBE cell growth is stimulated by treatment with a variety of growth factors that increase
mitogen-activated protein (MAP) activity. In this study, we examined
MAP kinase-dependent pathways as potential targets of
retinoid signaling and the role of MAP kinases in retinoid-induced
c-fos gene regulation. All-trans-retinoic acid
(t-RA) inhibited Jun N-terminal kinase (JNK) and, to a lesser extent,
extracellular signal-regulated kinase activity in normal HBE cells.
t-RA reduced c-fos mRNA and protein levels by
decreasing c-fos gene transcription. The c-fos
promoter was activated by co-transfection with a constitutively active
JNK kinase (SEK)-1 and suppressed by a dominant negative JNK kinase
kinase (MEKK)-1. Furthermore, c-fos expression was
inhibited by agonists of retinoic acid receptors (RARs) or retinoid X
receptors (RXRs), and suppression of c-fos promoter
activity by t-RA was abrogated by treatment with antagonists of RAR-
or of all the RXRs. These findings provide the first evidence that t-RA
inhibits JNK activity and demonstrate a potential role of
JNK-dependent pathways in the suppression of
c-fos expression by t-RA. Furthermore, c-fos
expression was inhibited through activation of RAR- and
RXR-dependent signaling pathways. In light of the growth
activation induced by JNK/SEK-dependent pathways in a
variety of cells, these data support further investigation into the
role of JNK-dependent signaling in the growth-suppressive effects of retinoids.
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INTRODUCTION |
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Retinoids control normal tracheobronchial epithelial growth and differentiation. Rodents that are deprived of vitamin A develop squamous metaplasia in the tracheobronchial epithelium, and normal epithelial differentiation is restored by vitamin A supplementation (1, 2). In tissue culture, human bronchial epithelial (HBE)1 cells undergo squamous differentiation with a variety of agents, and all-trans-retinoic acid (t-RA) inhibits this process (3-7). HBE cells treated with t-RA develop mucociliary features in collagen gels (3, 8). Grown in monolayer cultures, retinol-treated HBE cells undergo growth arrest with no evidence of morphologic differentiation (9).
Retinoids are ligands for the retinoic acid receptors (RAR-, -
,
-
) and retinoid X receptors (RXR-
, -
, -
) (reviewed in Ref.
10). RXRs form homodimers and heterodimers with RAR and a variety of
other nuclear hormone receptors, which are transcriptionally activated
by ligand binding. Of the known naturally-occurring retinoids, t-RA and
9-cis-retinoic acid activate RAR:RXR heterodimers, and
9-cis-retinoic acid also activates RXR homodimers (11, 12). Recently, synthetic retinoids have been designed that function as
selective agonists or antagonists of RARs or RXRs (13, 14). RAR- and
RXR-selective agonists can activate distinct signaling pathways and
induce different biologic effects (15-17), demonstrating divergent
roles for RAR- and RXR-dependent pathways.
The signaling pathways that mediate the biologic effects of retinoids
in normal HBE cells have not been defined. Potential candidates are
receptor tyrosine kinase-dependent pathways, which stimulate the growth of normal HBE cells (9, 18). Previous studies have
shown that growth factor- and retinoid-dependent signaling
pathways act antagonistically. t-RA inhibits expression of the
epidermal growth factor (EGF) receptor in human trophoblast cells and
reduces the level of one of its ligands, transforming growth
factor-, in normal HBE cells and teratocarcinoma cells (19-21).
Overexpression of transforming growth factor-
in teratocarcinoma cells antagonizes the growth inhibitory effects of t-RA, and
overexpression of the EGF receptor in HL-60 cells blocks the
cytodifferentiating effects of t-RA (22, 23). The EGF receptor
activates signaling through mitogen-activated protein (MAP) kinase
cascades, including the p42 and p44 (extracellular signal-regulated
kinase; ERK) kinases, the c-Jun N-terminal kinases
(JNK)/stress-activated protein (SAP) kinases, and the p38 kinase
(reviewed in Refs. 24 and 25). Activation of these kinase pathways
phosphorylates Elk-1, which is a component of the ternary complex that
includes the serum response factor, SAP-1, and SAP-2, leading to
increased Elk-1 DNA binding, ternary complex formation, and
transcriptional activation (26). Through this mechanism, EGF treatment
activates the Ets/serum response element motif in the c-fos
promoter, increasing c-fos expression (27, 28).
In this study, we examined MAP kinase-dependent pathways as potential targets of retinoid signaling and the role of MAP kinases in retinoid-induced c-fos gene regulation. t-RA inhibited JNK activity and reduced c-fos mRNA and protein levels by decreasing c-fos gene transcription. The c-fos promoter was activated by JNK-dependent pathway stimulation and inhibited by agonists of RARs or RXRs. These findings provide evidence that t-RA inhibited JNK activity and demonstrate a potential role of JNK-dependent pathways in the suppression of c-fos expression by t-RA. Furthermore, t-RA suppressed c-fos expression through activation of RAR- and RXR-dependent signaling pathways.
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EXPERIMENTAL PROCEDURES |
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Cells and Culture Conditions-- Normal HBE cells were cultured from bronchial mucosal biopsy samples taken from fresh surgical specimens as monolayer cultures on standard plasticware in keratinocyte serum-free medium (Life Technologies, Inc., Gaithersburg, MD) containing bovine pituitary extract and EGF as described previously (29). The MEK-1 inhibitor PD98059 was purchased from Calbiochem. For experiments involving RNA or protein preparation, cells were seeded on 10-cm plates at a density of 105/plate. For transient transfection assays, cells were seeded on 6-well plates at a density of 2 × 105/well. Retinoid treatment was begun the day after cell seeding.
Retinoids--
t-RA was purchased from Sigma. The RAR selective
agonist
(E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphth-alenyl)propenyl]benzoic acid (TTNPB/Ro13-7410) (30), the RAR-selective antagonist
(E)-6-[1-(4-carboxyphenyl)propen-2-yl]-4,4-dimethyl-3,4-dihydro-2H-1-benzothiopyran-1,1-dioxide (LGD100629/Ro41-5253) (31), and the RXR-selective antagonist (2E,4E,6Z)-3-methyl-6-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-3-propyl-oxy-2-naphthalenyl)-2,4,6-hepta-trienoic acid (LGD100754) (32) were generous gifts from Ligand Pharmaceuticals, San Diego, CA. The RXR-selective agonist
2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2-(4-carboxyphenyl)-1,3-oxathiolane (SR11235) was prepared as described (33).
Western Blot Analysis-- Whole cell lysates were prepared in MEGA-RIPA buffer as described previously (29). Protein lysate (50 µg) was separated by electrophoresis on a SDS-7.5% polyacrylamide gel, transferred onto a BA-S-83-reinforced nitrocellulose membrane (Schleicher & Schuell, Inc., Keene, NH), and immunoblotted overnight at 4 °C with a primary monoclonal antibody to c-fos, JNK-1, or ERK-1 and ERK-2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Binding was detected by using the ECL kit (Amersham, Inc.) according to the manufacturer's directions.
Northern Blot Analysis--
Total cellular RNA was prepared from
normal HBE cells, electrophoresed (20 µg/lane) on a 1% agarose gel
containing 2% formaldehyde, transferred to a nylon membrane
(Zetaprobe, Bio-Rad, Inc.), hybridized to an
[-32P]dCTP-labeled cDNA probe, washed, and
autoradiographed as described previously (29). The human
c-fos cDNA was obtained from Dr. Jeff Holt (Vanderbilt
University, Nashville, TN).
Reporter Plasmids and Expression Vectors--
The human
c-fos promoter from 327 to +40 (a
PstI-PstI genomic fragment which was a gift from
Dr. Jaideep Chaudhary, University of California-San Francisco) was
subcloned in sense (fos-LUC) and antisense (sof-LUC) orientation into
the pGL3-basic luciferase reporter plasmid (Promega, Inc.). The
constitutively active mutant cDNAs for SEK-1 (ED mutant, under the
control of the elongation factor promoter) and MEK-1 (R4F mutant, under
the control of the CMV promoter) were gifts from Dr. John M. Kyriakis
(Harvard Medical School, Boston, MA) and Dr. Natalie G. Ahn (University
of Colorado, Boulder, CO), respectively (34, 35). The dominant negative mutant cDNA for MEKK-1 under the control of the SV40 promoter was a
gift from Dr. F. X. Claret (University of California, San Diego)
(36). SV40-driven expression vectors were purchased; these contained
the DNA-binding domain of GAL-4 alone (GAL-DBD) or fused to the
transcription activation domain of Elk-1 (Elk-GAL-DBD) or
c-jun (JUN-GAL-DBD) (Stratagene, Inc., La Jolla, CA).
GAL-UAS-LUC is a luciferase reporter plasmid containing five repeats of
the GAL4 binding element (Stratagene).
Transient Transfection Assays-- The day after seeding, the cells were transfected with luciferase reporter plasmid (2 µg/well) for 6 h using LipofectAMINE (Life Technologies, Inc.). The transfectant solution was removed and the cells were treated with retinoids for 24 h. The cells were subjected to luciferase assays as described previously (29). Luciferase activities were expressed as the means and standard deviations of five identical wells and were normalized to cell number (2 × 105 cells/well).
JNK and ERK Assays--
Cells were treated for different periods
of time with t-RA (106 M). At the completion
of treatment, cells were harvested in WCEB buffer (25 mM
Hepes pH 7.7, 0.3 mM NaCl, 15 mM
MgCl2, 0.2 mM EDTA, 0.1% Triton X, 10 mM dithiothreitol, 0.25 mM sodium vanadate, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 2 mg/ml pepstatin, and 1 mM benzamidine), rotated at 4 °C for 20 min, and
centrifuged to pelletize cell debris. JNK-1 or ERK-1 and -2 were
immunoprecipitated from 100 µg of cell extract diluted in 200 µl of
WCEB and 600 µl of HBB buffer (20 mM Hepes pH 7.7, 50 mM NaCl, 0.1 mM EDTA, 2.5 mM
MgCl2, 0.05% Triton X, and phosphatase inhibitors) by
adding goat antibodies that recognize JNK-1 or ERK-1 and -2 (Santa Cruz Biotechnology), rotated at 4 °C for 2 h, and immunoprecipitated with 20 µl of protein G- and A-agarose beads. The beads were washed several times in ice-cold HBB and centrifuged. The immunoprecipitated proteins were resuspended in kinase buffer (20 mM Hepes pH
7.5, 20 mM glycerol phosphate, 10 mM
p-nitrophenyl phosphate, 10 mM MgCl2, 10 mM dithiothreitol, 50 mM
sodium vanadate, 20 µM ATP, and 0.1 mM
[
-32P]ATP (2000 cpm/pmol)), and 1 µg of GST-c-Jun
(1-179) (Santa Cruz) or myelin basic protein (Santa Cruz) were added.
The kinase reaction was performed at 30 °C for 20 min. The samples
were suspended in Laemmli buffer, boiled for 5 min, and the samples
were electrophoresed on acrylamide-SDS gels and autoradiographed.
Nuclear Run-on Analysis--
Cells were treated with media alone
or t-RA (106 M) for 6 h. Cells were
lysed in hypotonic buffer (0.8% Nonidet P-40, 10 mM Hepes
pH 7.9, 10 mM KCl, 0.75 M spermidine, 0.15 M spermine, 0.1 mM EDTA, pH 8.0, 0.1 mM EGTA, 10 mM dithiothreitol) using a Dounce homogenizer on ice, and centrifugation at 1,500 rpm. The pellets (nuclei) were resuspended thoroughly in transcription buffer (150 mM KCl, 5 mM MgCl2, 1 mM MnCl, 20 mM Hepes pH 7.9, 10% glycerol). Radioactive RNA was synthesized from the nuclei by adding the nucleotide mixture (transcription buffer containing 5 mM
dithiothreitol, 1 mM ATP, 1 mM GTP, 1 mM CTP, 250 µCi of [
-32P]UTP) and
incubating at 25 °C for 30 min. The nuclei were centrifuged at 1,500 rpm for 6 min, resuspended in DNase I buffer (2 mM Tris, pH
7.5, 5 mM MgCl2), and treated with 5 µl of
DNase I at room temperature for 10 min to digest genomic DNA. Nuclear
protein was digested by the addition of 5 µl of proteinase K, 500 µl of 1% SDS, 1 × TE, 10 µl of 0.5 M EDTA, and
240 µl of 1 × TE and incubated at 40 °C for 30 min. RNA was
prepared from the nuclear lysates by phenol/chloroform extraction,
NH3OAc/EtOH precipitation overnight at
20 °C,
centrifugation at 14,000 rpm for 10 min at 4 °C, resuspension of the
pellet in DNase I buffer, and DNase I digestion as described above.
Proteinase K treatment was repeated followed by phenol/chloroform
extraction, NH3OAc/EtOH precipitation. The RNA pellet was
resuspended in water, and incorporation of 32P was
quantitated with a scintillation counter. The c-fos and glyceraldehyde-3-phosphate dehydrogenase cDNAs and the
c-fos expression vector with no insert (negative control)
were immobilized on nylon membranes (5 µg/mm2) by
slot-blot and prehybridized (5 × SSPE, 5 × Denhardt's
solution, 0.5% SDS, 50% deionized formamide, 200 mg/ml tRNA, and 500 mg/ml single-stranded DNA) at 42 °C for 2 h. The RNA was boiled
for 3 min, chilled on ice, added to hybridization solution (the same as
the prehybridization solution) in sufficient quantity to cover the
slot-blot membrane at a concentration of 6.6 × 107
cpm/ml, and incubated at 42 °C for 48 h. The membranes were
washed to a stringency of 0.1 × SSC, 1% SDS at 65 °C and
autoradiographed.
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RESULTS |
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We examined the effect of t-RA treatment on MAP kinase activity in normal HBE cells. ERK and JNK activity were detected in normal HBE cells, and t-RA inhibited the activity of JNK and, to a lesser extent, ERK (Fig. 1A). Densitometric analysis revealed that at 24 h of t-RA treatment, JNK and ERK activities were 11 and 71%, respectively, of untreated cells. Transient co-transfection experiments were performed using reporter plasmids containing GAL4-response elements (GAL-UAS-LUC) and expression vectors containing the GAL4-DBD fused with the transcriptional activating domain of Elk-1 (Elk-GAL-DBD) or c-jun (Jun-GAL-DBD). GAL-UAS-LUC was activated by Elk-GAL-DBD and Jun-GAL-DBD, and t-RA inhibited 40% of the activity induced by Jun-GAL-DBD but not Elk-GAL-DBD (Fig. 1B). These findings demonstrate a prominent inhibition of JNK activity by t-RA treatment.
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ERK- and JNK-dependent signaling pathways activate
c-fos expression (27, 28). Therefore, we examined changes in
c-fos levels with t-RA treatment. c-Fos protein
levels were inhibited by t-RA in normal HBE cells (Fig.
2A). Moreover, t-RA decreased c-fos mRNA levels both time and dose dependently (Fig.
2, B and C). The effect of t-RA on
c-fos gene transcription was examined. Nuclear run-on
analysis revealed that t-RA decreased c-fos gene transcription (Fig. 3A).
Transient transfection assays with reporter plasmids containing the
c-fos promoter fragment from 327 to +40 in sense (fos-LUC)
or antisense (sof-LUC) orientation demonstrated that t-RA
sup- pressed fos-LUC activity, and this effect was dependent on
t-RA dose (Fig. 3B). These findings reveal that t-RA
suppressed c-fos gene transcription through proximal
c-fos promoter elements.
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The role of MAP kinase-dependent pathways in the regulation of c-fos promoter activity was examined. Transient co-transfection assays with fos-LUC and expression vectors containing constitutively active mutant MEK-1 or JNK kinase (SEK)-1 revealed that fos-LUC was minimally activated by MEK-1 and to a greater extent by SEK-1 (Fig. 4A). Treatment of normal HBE cells with the MEK-1 inhibitor PD98059 suppressed ERK activity but had no measurable effect on fos-LUC activity (data not shown). Co-transfection of an expression vector containing a dominant negative mutant JNK kinase kinase (MEKK)-1 inhibited fos-LUC activity (Fig. 4B). Assays of JNK and ERK activities in normal HBE cells transiently transfected with the dominant negative mutant MEKK-1 expression vector revealed selective inhibition of JNK activity (Fig. 4C). These findings support a role for JNK-dependent pathways in the regulation of c-fos promoter activity.
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Retinoids induce signaling events through nuclear receptor activation.
The potential roles of RAR- and RXR-dependent pathways were
investigated in the suppression of c-fos expression. Normal HBE cells express all RAR and RXR gene family members (29). Treatment
with RAR- (TTNPB) or RXR (SR11235)-selective agonists inhibited
c-fos mRNA levels and activation of the c-fos
promoter (Fig. 5, A and
B), suggesting that activation of RAR- and
RXR-dependent pathways inhibited c-fos
expression through suppression of proximal c-fos promoter
elements. Furthermore, treatment with antagonists of RAR- (LG100629)
or of all the RXRs (LG100754) partially abrogated the inhibition of
c-fos promoter activity by t-RA (Fig. 5C),
providing evidence that t-RA inhibited c-fos promoter
activity through RAR- and RXR-dependent signaling
pathways.
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DISCUSSION |
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In this study, we examined the effect of t-RA treatment on MAP kinase-dependent signaling pathways and the role of these pathways in t-RA-induced c-fos gene regulation. Our findings provide the first evidence that t-RA inhibits JNK and, to a lesser extent, ERK activity. Furthermore, we showed that t-RA suppressed c-fos mRNA and protein levels through a reduction in c-fos gene transcription. Previous studies have shown that t-RA inhibits c-fos gene transcription (37, 38), but the signaling pathways through which t-RA mediates this effect have not been examined. The c-fos promoter is activated by both ERK- and JNK-dependent pathways through activation of the ternary complex factor (27, 28). Consistent with these findings, we observed activation of the c-fos promoter by both MEK- and JNK-dependent pathways. Furthermore, inhibition of JNK-dependent pathways by transfection of a dominant negative mutant JNK kinase kinase (MEKK)-1 was sufficient to suppress c-fos promoter activity. These findings support the hypothesis that JNK inhibition contributes to retinoid-induced suppression of c-fos expression. While the suppression of JNK activity was more profound, ERK inhibition may also contribute to the inhibition in c-fos expression in t-RA-treated normal HBE cells.
Retinoid actions are mediated through nuclear receptor activation. RAR-
and RXR-selective retinoids have distinct biologic effects on cells in
tissue culture (39), suggesting that they activate different signaling
pathways. Consistent with this notion, RAR- and RXR-selective retinoids
can regulate the expression of distinct target genes or have opposing
effects on the same target gene (40, 41). Interestingly, both RAR- and
RXR-selective agonists inhibited c-fos expression in this
study, suggesting that RAR- and RXR-dependent pathways
share certain signaling events. Supporting this possibility, both RAR-
and RXR-selective agonists inhibit the squamous differentiation of HBE
cells (42). This appears to be at least partly the result of the
suppressive effects of these retinoids on the AP-1 transcription
factor, which binds to the AP-1 sites in the promoters of a variety of
genes required for the induction of HBE squamous differentiation,
activating their expression (42). Furthermore, we found that an RAR-
antagonist abrogated the effect of t-RA on the c-fos
promoter, supporting a role for RAR-
in the inhibition of
c-fos expression. In addition to reducing c-fos
expression, RAR-
also activates the expression of transforming
growth factor-
and insulin-like growth factor-binding protein-3 in
normal HBE cells (43). Unexpectedly, an RXR antagonist that blocks
activation of RXR homodimers abrogated the inhibition of
c-fos promoter activity by t-RA. t-RA does not
transcriptionally activate RXRs (reviewed in Ref. 10). A potential
explanation for this finding is that t-RA can undergo
stereoisomerization intracellularly to 9-cis-retinoic acid
(44) and thus activate RXR-dependent signaling, which the
RXR antagonist would block.
Mechanisms by which retinoid receptors suppress c-fos
expression have not been elucidated. Retinoid receptors modulate gene expression directly by binding to response elements within gene promoters and indirectly by binding to other transcription factors. The
c-fos promoter from 327 to +40 (45) contains a variety of
response elements that regulate its activity, including binding sites
for cAMP response element-binding protein, STAT factors, YY-1, AP-1,
and the ternary complex, but there are no consensus retinoid response
elements (AGGTCA repeats separated by 1, 2, or 5 nucleotides),
suggesting that retinoid receptors do not bind directly to this portion
of the c-fos promoter. We demonstrated a potential role for
JNK-dependent pathways in the suppression of
c-fos expression by t-RA, supporting an indirect interaction of retinoid receptors with the c-fos promoter through
JNK-dependent signaling pathways. Interactions between
retinoid receptors and MAP kinase pathways have been not described. The
estrogen receptor is phosphorylated through MAP
kinase-dependent pathways, enhancing estrogen receptor
transcriptional activity (46), which provides a precedent for
investigating interactions between retinoid receptors and components of
MAP kinase signaling pathways.
Normal HBE cells secrete a variety of peptide growth factors that stimulate growth through autocrine and paracrine mechanisms (9, 18). It is possible that the growth of normal HBE cells is dependent upon MAP kinase activation by endogenous and exogenous growth factors. This notion is supported by the observation that withdrawal of EGF or bovine pituitary extract, which contains a number of peptide growth factors, from the media attenuates the growth of normal HBE cells (9). We and others previously showed that treatment with t-RA inhibits normal HBE cell growth, and interruption of an autocrine growth stimulus contributed to the growth inhibition induced by t-RA (9, 29). Inhibition of JNK or other components of growth factor-dependent signaling pathways may be an important mechanism by which retinoids inhibit the growth of normal HBE cells, and future work will examine this possibility.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants R29 CA67353 and P50 CA70907 (Lung Cancer SPORE).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Box 80, M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030.
1 The abbreviations used are: HBE, human bronchial epithelial; EGF, epidermal growth factor; t-RA, all-trans-retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; JNK, c-Jun N-terminal kinase; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; SAP, stress-activated protein kinase; CMV, cytomegalovirus.
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REFERENCES |
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