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Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 321-332
Two Candidate Downstream Target Genes for E2A-HLF
By
Hidemitsu Kurosawa,
Kumiko Goi,
Takeshi Inukai,
Toshiya Inaba,
Kun-San Chang,
Tetsuharu Shinjyo,
Karen M. Rakestraw,
Clayton W. Naeve, and
A. Thomas Look
From the Department of Experimental Oncology and the Center for
Biotechnology, St Jude Children's Research Hospital, Memphis, TN; the
Department of Pediatrics, University of Tennessee College of Medicine,
Memphis, TN; Jichi Medical School, Tochigi, Japan; and the Division of
Laboratory Medicine, the University of Texas M.D. Anderson Cancer
Center, Houston, TX.
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ABSTRACT |
The E2A-HLF fusion gene, formed by the t(17;19)(q22;p13)
chromosomal translocation, is thought to drive the leukemic
transformation of early B-cell precursors by repressing an
evolutionarily conserved apoptotic pathway. To test this hypothesis, we
sought to identify downstream targets of E2A-HLF in
t(17;19)+ pro-B leukemia cells (UOC-B1) that had been
transfected with a zinc-inducible vector encoding a dominant-negative
suppressor (E2A-HLF[dn]) of the oncoprotein. Representational
difference analysis of mRNAs from E2A-HLF(dn)+ UOC-B1
cells grown with (E2A-HLF inactive) or without (E2A-HLF active) the
addition of zinc yielded several differentially expressed cDNA
fragments that were individually subcloned. Two of the clones, designated F-5 and G-4, hybridized with mRNAs that were upregulated by
E2A-HLF. Levels of both transcripts declined sharply within 8 to 12 hours after suppression of E2A-HLF DNA-binding activity, becoming
undetectable after 96 hours. The F-5 cDNA was identified as a portion
of ANNEXIN VIII, whose product was expressed in promyelocytic leukemia cells and UOC-B1 cells, but not in other leukemic cell lines.
A novel full-length cDNA cloned with the G-4 fragment encoded a protein
that we have named SRPUL (sushi-repeat protein upregulated in
leukemia). It is normally expressed in heart, ovary, and placenta, but
could not be detected in leukemic cell lines other than UOC-B1. Neither
protein prevented apoptosis in interleukin-3-dependent murine pro-B
cells, suggesting that they have paraneoplastic roles in leukemias that
express E2A-HLF, perhaps in the disseminated intravascular coagulopathy
and hypercalcemia that characterize these cases.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE E2A-HLF FUSION gene is
generated by the t(17;19) (q23;p13) in cases of pro-B acute leukemia
that occur in older children and adolescents.1,2 The
leukemias associated with this genetic alteration do not respond well
to standard chemotherapy and typically present with an intravascular
coagulopathy and hypercalcemia unusual findings in children with
B-cell precursor acute lymphoblastic leukemia
(ALL).3,4 The encoded E2A-HLF protein contains
the AD1 and AD2 transactivation domains of E2A,5-8 linked
to the bZIP DNA binding/protein dimerization region of HLF. HLF is a member of the PAR subfamily of leucine zipper-type transcription factors that is normally expressed in liver, kidney cells, and neurons
within the central nervous system.1,2,9 Overexpression of a
dominant-negative inhibitor of the chimera in leukemic cells transformed by E2A-HLF results in programmed cell death.10
Similarly, introduction of wild-type E2A-HLF into Baf-3 interleukin-3
(IL-3)-dependent murine pro-B lymphocytes allows the cells to survive
in G1 phase of the cell cycle for 2 weeks or longer after IL-3
withdrawal, and to resume exponential growth when IL-3 is
reintroduced.10 These findings suggest that E2A-HLF
contributes to leukemogenesis by altering the transcriptional control
of genes responsible for lineage-specific cell suicide in pro-B cells,
leading to the survival of defective cells that normally would be
eliminated by apoptosis.10
A critical first step in understanding malignant transformation
mediated by E2A-HLF is to identify downstream responder genes targeted
by this oncoprotein. Thus, we are exploiting a matched cell system
based on the presence or absence of E2A-HLF transcriptional activity.
It uses a t(17;19)+ pro-B leukemia cell line (UOC-B1)
transduced with a zinc-inducible vector encoding a dominant-negative
suppressor of E2A-HLF, which sequesters the chimeric transcription
factor within nonfunctional complexes, thereby blocking its ability to
bind to the HLF DNA consensus sequence.10 Of
several possible approaches to identify potential targets of
E2A-HLF action, we have chosen the subtractive process of
representational difference analysis (RDA), a polymerase chain reaction
(PCR)-based technique that has proved useful for selecting and cloning
cDNAs that correspond to differentially expressed
mRNAs.11-15
Here we describe the use of RDA to identify two genes whose expression
depends on the activity of E2A-HLF. One gene was identified as
ANNEXIN VIII, which is also expressed in acute promyelocytic leukemia.16,17 The second is a novel gene that encodes a
protein we have named SRPUL (sushi-repeat protein upregulated in
leukemia), which contains consensus repeat motifs found in the
extracellular domain of members of the selectin
family.18,19 SRPUL is most closely related to a
gene called SRPX (sushi repeat-containing protein), or
ETX1, which may be involved in X-linked retinitis pigmentosa.20,21 The ANNEXIN VIII and SRPUL proteins could not substitute for E2A-HLF in promoting the survival of murine pro-B
cells deprived of growth factor, suggesting that they are more likely
to be involved in paraneoplastic syndromes characteristic of
t(17;19)+ leukemias than in processes leading to malignant
transformation.
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MATERIALS AND METHODS |
Construction of eukaryotic expression vectors.
Expression plasmids containing wild-type E2A-HLF, a
dominant-negative suppressor of E2A-HLF binding
(E2A-HLF[dn]), ANNEXIN VIII, and SRPUL-CF
were constructed in the pMT-CB6+ eukaryotic expression vector (a gift
from Dr F. Rauscher III, Wistar Institute, Philadelphia, PA). This
vector contains the inserted cDNA under control of the sheep
metallothionine promoter, as well as the neomycin-resistance gene
(neoR) driven by the SV40 early promoter.
Cell culture and cell survival assay.
UOC-B1 human pro-B-cell leukemia cells that express E2A-HLF and
HL-60 human myeloid leukemia cells were cultured in RPMI 1640 medium
containing 10% fetal calf serum. UOC-B1(dn) clone 3 and control UOC-B1
(pMT) cells transfected with either E2A-HLF(dn) or the pMT
empty vector were prepared as described previously.10 E2A-HLF(dn) expression was induced in the UOC-B1(dn)3 cells by treating them with 100 µmol/L ZnSO4 for 24 hours. Viable
cell counts were determined by trypan-blue dye exclusion in triplicate assays. FL5.12 pro-B lymphocytes22 were cultured in
RPMI-1640 medium containing 10% fetal calf serum and 10%
WEHI-3B-conditioned medium (as a source of IL-3). Transfectants were
generated by electroporation using 2 × 107 cells and
80 µg of plasmid DNA with a gene pulser (Bio-Rad, Hercules, CA) set
to deliver 320 V and 960 µF. The cells were then cultured in 24-well
dishes and selected in the presence of the G418 neomycin analogue (0.6 mg/mL) for 2 to 4 weeks. The levels of protein expression were
determined by Western blotting, and cell clones with regulated expression were selected for further experimentation. For cell survival
assays, protein expression was induced by treating cells with 100 µmol/L ZnSO4 for 16 hours before growth factor
deprivation. IL-3 was removed by repeated centrifugation in fresh
media, and the cells were adjusted to 5 × 105 per mL
on day 0 and cultured without IL-3. Viable cell counts were determined
by trypan-blue dye exclusion.
Northern blot analysis.
One microgram of messenger RNA, extracted with Fast Track (Invitrogen,
Carlsbad, CA), was separated by electrophoresis in 1% agarose gels
containing 2.2 mol/L formaldehyde and transferred to nylon membranes.
Signals were visualized by autoradiography after hybridization to a
human HLF, ANNEXIN VIII, SRPUL, or  -ACTIN 32P-labeled cDNA. A multiple-tissue Northern blot
(Clontech, Palo Alto, CA) contained 2 µg each of poly(A) RNAs
isolated from various normal human tissues.
Reverse transcriptase (RT)-PCR.
Total RNA was extracted from leukemia cells using the Trizol reagent
(GIBCO-BRL, Gaithersburg, MD), following the manufacturer's directions. Reverse transcription was performed with 2 µg of total RNA. RNA was resuspended in a final volume of 11 µL with 50 ng of
random hexamer primer (Pharmacia Biotech, Uppsala, Sweden), incubated
at 80°C for 5 minutes and cooled on ice. Reverse transcription was
performed using 200 U SuperScript II reverse transcriptase (GIBCO-BRL)
in the manufacturer's buffer in the presence of 0.5 mmol/L dNTP and
1.0 mmol/L dithiothreitol in a final volume of 20 µL, and incubated
at 42°C for 1 hour. Each sample was then incubated with 1 µL of
RNaseH (GIBCO-BRL) at 37°C for 20 minutes. As a negative control,
the reaction was also performed for each sample without reverse
transcriptase. cDNA samples were stored at  20°C. PCR was
performed using a DNA Thermal cycler (Perkin Elmer, Applied Biosystems,
Inc, Foster City, CA). PCR reactions contained 1 µL of the cDNA
product, 1× reaction buffer, 1.5 mmol/L MgC12, 0.2 mmol/L dNTP, 10 pmol of each primer, and 2.5 U of Taq polymerase
(Perkin Elmer) in a total volume of 50 µL. Thermocycling parameters
for the first PCR and the sequences of the specific primers are as
follows: E2A-HLF, 30 cycles at 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 1 minute; primers described previously1; ANNEXIN VIII, 40 cycles at 94°C for 30 seconds, 52°C for 30 seconds, and 72°C for 1 minute; sense
primer 5 -ATGCAGAGACCCTCTACAAA-3 and antisense primer
5-CATAGTCTTCCTCATACGCC-3; SRPUL, 40 cycles at 94°C
for 30 seconds, 52°C for 30 seconds, and 72°C for 1 minute; sense primer 5-GAGGAAATTATCACAGCAGC-3 and antisense
primer 5-CAGTGTAACGAATCACATGC-3; c-ABL, 30 cycles at
94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute; sense primer 5-GTATCATCTGACTTTGAGCC-3 and
antisense primer 5-GTACCAGGAGTGTTTCTCCA-3. A second PCR was performed for SRPUL using the same primers and 3 µL of the first
PCR products as template for 30 cycles. The amplification of c-ABL mRNA
was performed as a control to assess the quality of each RNA sample.
These sets of the primers amplify a 342-bp ANNEXIN VIII product, a
501-bp SRPUL product, and a 288-bp c-ABL product. Ten microliters of
the PCR products were electrophoresed in a 2.0% agarose gel and
transferred to a nylon membrane (NEN Life Science, Boston, MA) with 0.4 N NaOH. The membranes were hybridized with an internal probe
end-labeled with 32P using polynucleotide kinase, and
visualized by autoradiography. The sequences of the internal probes
were as follows: E2A-HLF, 5-ACCCTCCCTGACCTGTCTCG-3;
ANNEXIN VIII, 5-CTCATTGTGGCCCTTATGTA-3; SRPUL,
5-GAGAAATTGACTGCTCGAGT-3; c-ABL,
5-TAACTAAAGGTGAAAAGCTCC-3.
Immunoblot analysis.
Cells were solubilized in Nonidet P-40 (NP-40) lysis buffer (150 mmol/L
NaCl, 1.0% NP-40, 50 mmol/L Tris [pH 8.0]), and total cellular
proteins were separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). After wet electrotransfer of the products
onto nitrocellulose membranes, immunoblotting was performed with
ANNEXIN VIII17 and HLF(C)23 specific
rabbit antiserum, preimmune rabbit serum, and FLAG
monoclonal antibody (MoAb) (M2; Eastman Kodak, New Haven,
CT). The blots were then stained with primary antibodies and studied by
horseradish peroxidase-conjugated donkey anti-rabbit- and sheep
anti-mouse-enhanced chemiluminescence (Amersham Life Sciences Inc,
Arlington Heights, IL).
RDA.
RDA was performed essentially as described by Hubank and
Schatz.11 Briefly, mRNAs were isolated from UOC-B1(dn)3
cells harboring the E2A-HLF(dn) construct and grown in the
presence or absence of 100 µmol/L zinc in the culture medium. A
10-µg sample of mRNA from each cell population was used for RDA
analysis. cDNAs were synthesized from the mRNAs and digested with
DpnII. Adapters, 5-AGCACTCTCCAGCCTCTCACCGCA-3
(RBgl24) and 5-GATCTGCGGTGA-3 (RBgl 12), were ligated to
the DpnII-digested cDNA. This mixture was amplified by PCR with
RBgl 24 oligonucleotides, and the adapters were excised with
DpnII. A second pair of adapters,
5-ACCGACGTCGACTATCCATGAACA-3 (JBgl 24) and
5-GATCTGTTCATG-3 (JBgl 12), were ligated to the amplified
fragments from UOC-B1(dn) cells grown without zinc (tester) and
hybridized with the RBgl 24-amplified cDNA fragments (RBgl adapters
removed) from zinc-induced UOC-B1(dn) cells (driver) at a ratio of 1:80
for 20 hours. The hybridization mix was used as template for
amplification by PCR. A second round of subtraction was performed by
removing the JBgl adapters from an aliquot of the first-round PCR
product, ligating a third pair of oligonucleotides adapters,
5-AGGCAACTGTGCTATCCGAGGGAA-3 (NBgl 24) and
5-GATCTTCCCTCG-3 (NBgl 12), and hybridizing with driver
amplicons at a ratio of 1:800. A third round of subtraction was
performed by removing the NBgl adapters from an aliquot of the
second-round PCR product, relegating the JBgl 24 and JBgl 12 adapters,
and hybridizing with driver amplicons at a ratio of 1:8000. After PCR
amplification with JBgl 24 primers, several bands were evident in 2.0%
agarose gels stained ethidium bromide; these were individually
subcloned and tested for differential expression by Northern blotting
of RNAs from UOC-B1(dn)3 cells grown in the absence or presence of zinc.
Cloning of ANNEXIN VIII full-length cDNA by RT-PCR.
The ANNEXIN VIII cDNA was cloned by RT-PCR using upstream
and downstream primers with flanking EcoRI sites
(5'-CGTGTGGAATTCCAGCAGAGGCCAACC-3' and
5'-CTCTGAATTC-ATGGTCTTTGCTCTTG-3'). RT-PCR was performed with a cDNA
Cycle Kit (Invitogen). The nucleotide sequence of the insert, confirmed
by DNA sequencing, was identical to that of the VAC cDNA.24
Metabolic labeling and immunoprecipitation.
Cells were metabolically labeled by incubation in methionine-free
medium for 30 minutes. [35S]methionine (New England
Nuclear, Boston, MA) was then added (0.5 mCi/mL of culture medium), and
the cells were incubated for an additional 60 to 180 minutes. After
removal of the medium, the cells were lysed for 60 minutes on ice in
radioimmunoprecipitation assay buffer (150 mmol/L NaCl, 1.0% NP-40,
0.5% SDS, 50 mmol/L Tris-HCl [pH 8.0]), plus 50 µg/mL leupeptin,
0.5% aprotinin, 1 mmol/L Na vanadate, 1 mmol/L phenylmethylsulfonyl
fluoride, and 2 mmol/L EDTA. The cell lysates from 2 × 107 cells were clarified by centrifugation at 20,000g
for 30 minutes. These lysates were then mixed with the designated
rabbit antiserum or  -FLAG MoAb for 60 to 120 minutes at 4°C, and
immune complexes were collected by incubation for 60 minutes with
protein A-Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden). To
detect secreted proteins, we labeled FL5.12 cells expressing SRPUL in 4 mL of methionine-free medium, which also was immunoprecipitated with the -FLAG MoAb and protein A-Sepharose. The proteins were analyzed by SDS-PAGE followed by autoradiography. The level of protein expression was quantified with use of a phosphorimager (Molecular Dynamics, Sunnyvale, CA).
Isolation, sequence analysis, and in vitro transcription-translation
of the SRPUL cDNA.
A  -Zap II human heart cDNA library (Stratagene, La Jolla, CA) and a
-Zap II UOC-B1(dn) cDNA library (Stratagene) were hybridized with
the 340-nucleotide G-4 probe. The pBluescript phagemid was purified
from positive clones by in vivo excision from the -Zap II vector,
using ExAssist helper phage (Stratagene). The full-length SRPUL
clone hybridized to the same E2A-HLF-regulated transcript in
UOC-B1(dn) cells as did the G-4 probe. Sequence analysis of the
SRPUL cDNA was performed by the Center for Biotechnology at St
Jude Children's Research Hospital using dye-terminator cycle sequencing ready reaction kits with AmpliTaq DNA polymerase FS (Perkin-Elmer [PE], Applied Biosystems, Inc [ABI]) and synthetic oligonucleotides. Samples were electrophoresed, detected, and analyzed
on PE/ABI model 373, model 373 Stretch, or model 377 DNA sequencers. In
vitro transcription-translation analysis of phagemid DNA (1.0 µg) was
performed with [35S]methionine (Amersham, translation
grade) in a coupled transcription-translation assay system (TNT Kit;
Promega, Madison, WI). Products were analyzed by electrophoresis in
SDS-PAGE, after which the gels were dried and subjected to
autoradiography.
Construction of plasmids expressing SRPUL with a C-terminal FLAG-tag
(SRPUL-CF).
We added the FLAG epitope, with the amino acid sequence DYKDDDDK, to
the C-terminus of the SRPUL protein. To generate
pMT-CB6+/SRPUL-CF, we amplified a sequence by PCR with
full-length SRPUL as a template that included the translation
initiation signal and FLAG coding sequence, using and an upstream
primer with a flanking HindIII site
(5-CCCAAGCTTGCCATGGCCAGTCAGC-TAACTCA-3) and a downstream primer with a flanking BamHI site
(5-CGCGGATCCTCACTTGTCATCGTCGTCCTTGTAGTCCTCGCATATGTCCCTTTGCT-3). PCR was performed using Pfu DNA polymerase (Stratagene). The PCR product was double-digested with HindIII and BamHI and
cloned into HindIII/BamHI-digested pMT-CB6+. The
nucleotide sequence of the insert was confirmed by DNA sequencing.
Indirect immunofluorescence.
Indirect immunofluorescence analysis was performed as described by
Murti et al,25 with minor modifications. Cells were fixed with 3.7% paraformaldehyde for 20 minutes, washed three times with
PBS, and permeabilized in 0.2% Triton-X (Sigma, St Louis, MO)/PBS
for 10 minutes at room temperature. After three washes with PBS, the slides were preincubated with 5% skim milk powder, and
1% normal horse serum in PBS for 30 minutes at room temperature, and
then incubated with -FLAG MoAb M2 (10 µg/mL) for 1 hour at room
temperature in a humidified chamber. The cells were washed four times
with PBS, and then incubated with fluorescein (FITC)-conjugated goat-mouse IgG (1/2,000) (Jackson Immuno Research Laboratories, West
Grove, PA) for 30 minutes at room temperature. The samples were
examined with a Nikon Labophot-2 microscope equipped with epifluorescence optics for FITC detection and
4.6-diamidino-2-phenylindole (DAPI).
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RESULTS |
Identification of genes regulated by E2A-HLF.
The effects of blocking E2A-HLF activity in UOC-B1 leukemia cells were
studied with a conditional dominant-negative mutant,10 E2A-HLF(dn), which lacks the AD1 transactivation domain of
E2A6,7 and contains a mutated HLF DNA-binding domain with
an intact leucine-zipper domain. Thus, E2A-HLF(dn) will heterodimerize
with E2A-HLF and sequester the endogenous oncogenic form of the protein
within inactive complexes that are unable to bind to DNA.10
In clones transfected with the E2A-HLF(dn) cDNA in a
zinc-inducible vector, expression of the mutant protein was highly
dependent on addition of zinc to the culture medium, as shown in
Fig 1 for UOC-B1(dn)3 cells. By contrast,
the endogenous E2A-HLF oncoprotein was expressed at constant levels in
both UOC-B1(dn)3 and control cells, regardless of the addition of zinc.
Immunoblot analysis showed that E2A-HLF(dn) levels were 10-fold higher
than those of the wild-type protein at 4 to 8 hours after the addition
of zinc, even though expression of the mutant mRNA was at lower levels than that of the wild-type E2A-HLF transcript
(Fig 2C). This likely reflected either
higher translation efficiency or a prolonged half-life of the mutant
compared with the wild-type protein. The mutant protein was relatively
stable, in that its levels did not decrease appreciably until 2 days
after zinc was removed from the cultures (Fig 1). Binding of E2A-HLF by
the E2A-HLF(dn) mutant, leading to protein heterocomplexes unable to
interact with the HLF consensus sequence, was shown in a previous
publication.10 In that study, the E2A-HLF(dn) protein
induced apoptosis of UOC-B1(dn) cells, beginning approximately 72 hours
after induction of its expression and 48 hours after the loss of
E2A-HLF DNA-binding activity. The rate of apoptotic death exceeded the
rate of mitotic division, resulting in a progressive loss of viable
cells over a 7-day period, with a gradual return to normal survival
after removal of zinc from the culture medium.10 Thus, our
inducible system provided matched cell populations with active and
suppressed E2A-HLF functions, allowing the analysis of differentially
expressed genes.
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| Fig 1.
Immunoblot analysis of E2A-HLF and E2A-HLF(dn) proteins.
Cell lysates were prepared from UOC-B1(dn)3 cells grown in the absence
of zinc (lane 1) or in its presence (100 µmol/L) for the indicated
intervals (lanes 2 through 7). After 24 hours, a portion of the cells
were washed to remove zinc and cultured for the indicated intervals in
the absence of the metal (lanes 8 through 11). Control UOC-B1 cells
electroporated with the pMT empty vector (UOC-B1[pMT]) and grown for
24 hours in medium without (lane 12) or with 100 µmol/L zinc (lane
13).
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| Fig 2.
Upregulation of ANNEXIN VIII and SRPUL
cDNAs by E2A-HLF. Northern blot analysis of poly(A) RNA (1 µg
per lane) prepared from UOC-B1(dn)3 cells grown in zinc-free
medium (lane 1) or in medium supplemented with zinc for the indicated
times (lanes 2 through 8), followed by zinc depletion (lanes 9 through
12). Control experiments were performed with UOC-B1 cells transfected
with an empty vector and grown in zinc-free medium (lane 13) or for 24 hours in the presence of zinc (lane 14). (A) The blot was
hybridized with ANNEXIN VIII (F-5) ( ), (B)
SRPUL (G-4) ( ), or (C) HLF cDNA fragments, and then
stripped and rehybridized with a -ACTIN probe (D) ( ).
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Using mRNAs from UOC-B1(dn)3 cells grown in the absence of zinc
(E2A-HLF transcriptionally active) or for 24 hours in its presence
(E2A-HLF transcriptionally inactive), we prepared cDNAs and subjected
them to three rounds of sequential hybridization and PCR amplification
according to the RDA protocol of Hubank and Schatz.11
Several differentially amplified cDNA fragments were individually
subcloned and tested for differential gene expression by Northern
blotting of UOC-B1(dn) RNAs. cDNA fragments representing two
differentially regulated genes were identified, and designated F-5 and
G-4. Sequence comparisons showed that the 351-nucleotide F-5 fragment
corresponded to part of the coding region and the 3
nontranslated region of ANNEXIN VIII, also known as human
vascular anticoagulant beta (VAC-).24 By contrast, the
G-4 cDNA fragment represented a novel gene, because it did not
correspond to any previously known cDNA identified with the BLAST
search utility (National Center for Biotechnology
Information). The predicted amino acid sequence of the protein
contained so-called sushi repeats,20,26 so we have named
this gene SRPUL, for sushi-repeat protein upregulated in
leukemia.
Northern blot analysis showed high levels of ANNEXIN VIII and
SRPUL mRNA expression in UOCB1(dn)3 cells, which decreased
rapidly, coincident with expression of the E2A-HLF(dn) mRNA (Fig 2).
The ANNEXIN VIII mRNA level declined fivefold by 8 hours after
the addition of zinc and 25-fold by 24 hours, becoming undetectable by
96 hours (Fig 2A). Similarly, there was a threefold decrease in
concentrations of SRPUL mRNA within 12 hours and a 60-fold deficit by 24 hours; SRPUL transcripts were also not detectable after 96 hours (Fig 2B). Removal of zinc from the growth medium restored both SRPUL and to a lesser extent ANNEXIN VIII
mRNA expression by 72 hours, coincident with a decline in the
E2A-HLF(dn) protein level (Fig 1). ANNEXIN VIII and
SRPUL were unaffected by zinc in control UOC-B1 cells
(UOC-B1[pMT]) lacking the dominant-negative construct, confirming
that the observed changes in gene expression were stimulated by E2A-HLF
(Fig 2, lanes 13 and 14).
To show ANNEXIN VIII regulation at the protein level, we used specific
antiserum to immunoprecipitate metabolically labeled lysates from
UOC-B1(dn) and UOC-B1(pMT) cells grown with or without zinc. ANNEXIN
VIII protein synthesis rates were threefold lower in UOC-B1(dn) cells
grown in the presence of zinc, and remained unchanged in
control UOC-B1(pMT) cells, regardless of the zinc concentration (Fig 3).
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| Fig 3.
Regulation of ANNEXIN VIII protein synthesis.
UOC-B1(dn)3 (lanes 1 and 2) or control UOC-B1(pMT) (lanes 3 and 4)
cells were grown without exogenous zinc (lanes 1 and 3) or for 24 hours
in the presence of zinc (100 µmol/L; lanes 2 and 4). Metabolically
labeled lysates of these cells were immunoprecipitated with an
ANNEXIN VIII antiserum.
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Expression of ANNEXIN VIII and SRPUL is associated with E2A-HLF
expression in early B-lineage leukemias.
To assess the linkage between expression of the E2A-HLF
chimeric oncogene and the expression of ANNEXIN VIII and
SRPUL, we performed RT-PCR analysis of leukemic cell lines and
cryopreserved primary leukemia patient samples with and without the
t(17;19). As shown in Fig 4, the UOC-B1 and
HAL-01 t(17;19)-positive cell lines, as well as two primary patient
leukemia samples (patients #1 and #21), each express the
E2A-HLF fusion transcript as well as mRNAs encoding ANNEXIN
VIII and SRPUL. The E2A-HLF fusion transcripts migrate differently for each of the patients shown in Fig 4, because the joining region at the fusion junction contains unique numbers of
inserted nucleotides, as described previously.1,2 The UOCB1
cell line was derived from cells of patient 1,1 which explains the identical pattern of E2A-HLF for these two samples. Examples of RNAs from three other leukemia cell lines lacking the
t(17;19) are shown in Fig 4, including the Nalm6 and 697 early B-lineage and the Jurkat T-cell cell lines. These cell lines did not
express either E2A-HLF or transcripts encoding ANNEXIN
VIII or SRPUL, although they did express the c-ABL
control, indicating that the RNA was intact for these samples. In all,
a total of 10 leukemic cell lines were tested using the RT-PCR assay
shown in Fig 4. ANNEXIN VIII expression was detected in the REH
early B-lineage and the U937 monocytic cell lines lacking
E2A-HLF, in addition to the four samples expressing E2A-HLF
shown in Fig 4. SRPUL was not expressed by any of the cell lines
evaluated except those shown in Fig 4 that expressed
E2A-HLF. Thus, our analysis suggests that
expression of ANNEXIN VIII and SRPUL is consistently associated with expression of the E2A-HLF fusion gene, and that expression of these responder genes is relatively rare in leukemias transformed through the auspices of other genetic alterations.
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| Fig 4.
RT-PCR analysis. Each RNA was transcribed with (+; odd
lanes) or without (; even lanes) RT. The PCR products were
transferred to a nylon membrane and analyzed by hybridization with
end-labeled internal oligonucleotide probes specific for each gene.
RNAs were evaluated for the UOC-B1 (lanes 1 and 2) and HAL-01 (lanes 3 and 4) t(17;19)-positive human leukemic cell lines, patient samples
with t(17;19)-positive ALL (lanes 5 through 8), the t(17;19)-negative
Nalm6 early B-lineage leukemic cell line (lanes 9 and 10), 697 pre-B
leukemic cell line (lanes 11 and 12), and Jurkat T-cell leukemic cell
line (lanes 13 and 14), as well as a control PCR reaction lacking RNA
template (lanes 15 and 16).
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Overexpression of E2A-HLF induces ANNEXIN VIII but not SRPUL
expression in HL-60 cells.
Next, we tested whether expression of the intact E2A-HLF chimeric
protein can induce expression of ANNEXIN VIII and SRPUL mRNAs.
For these experiments, HL-60 cells were transfected with a
pMT-CB6+/E2A-HLF construct to generate cell clones in which the
oncogenic form of E2A-HLF is regulated by the sheep metallothionein promoter (eg, HL-60[E2A-HLF]24 in Fig 5).
By 4 hours after addition of zinc to the culture medium, E2A-HLF
protein levels had increased fivefold (Fig 5A, lane 3) by comparison
with background levels mediated by the sheep metallothionine promoter
in the absence of zinc (lane 1). ANNEXIN VIII mRNA expression
lagged several hours behind the expression of E2A-HLF, becoming
detectable 8 hours after zinc addition and reaching its highest level
by 24 hours (Fig 5B). E2A-HLF did not induce SRPUL mRNA
expression in HL-60 cells (Fig 5C).
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| Fig 5.
ANNEXIN VIII expression in HL-60 cells engineered to
express E2A-HLF. (A) Immunoblot analysis of HL-60 cells transfected
with zinc-regulated pMT-CB6+/E2A-HLF (HL-60[E2A-HLF]24 cells). Cell
lysates were prepared from cells grown in medium lacking exogenous zinc
(lane 1) and at serial intervals after the addition of zinc (100 µmol/L) to the medium (lanes 2 through 8). Control HL-60 cells
transfected with the empty pMT vector (HL-60 [pMT]) did not express
E2A-HLF, whether cells were grown in the absence (lane 9) or presence
(lane 10) of zinc. Northern blot analysis of poly(A) RNA (1 µg per
lane) prepared from HL-60(E2A-HLF)24 or HL-60(pMT) cells grown under
the same conditions hybridized with either (B) ANNEXIN VIII or
(C) SRPUL cDNA probes. (D) The blot was stripped and
rehybridized with a control -ACTIN probe.
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Cloning of full-length SRPUL.
Northern blotting with the 340-bp G-4 probe showed a SRPUL mRNA
of 2.5 kb, which was expressed at highest levels in heart, ovary, and
placenta (Fig 6). Using the
G-4 probe to screen cDNA libraries from mRNA of human heart library and
UOC-B1(dn) cells, we obtained positive clones with approximately 2-kb
cDNA inserts. The longest cDNA consisted of 2,133 bp, and had one long
open-reading frame that extended from nucleotide 419 to 1813, potentially encoding a 465-amino acid protein (GenBank accession no.
AF060567). The initial ATG codon was preceded by an in-frame
termination codon, and hence constitutes the bona fide initiation codon
for this protein. The molecular weight of the protein was predicted to
be 53 kD, consistent with results of an in vitro
transcription-translation experiment showing a 50-kD product (data not
shown). Comparison of the deduced SRPUL amino acid sequence
with protein databases revealed three consensus (sushi) repeats of
approximately 60 amino acids, starting with amino acid 59 (Fig 7). These repeats contain six
conserved cysteines, and are characteristic motifs in members of the
selectin family of cell membrane proteins with functional roles in cell
adhesion.18,19 The highest homology scores (identity 47%,
similarity 67%) were obtained with a sushi repeat-containing protein
designated SRPX or ETX1,20,21 which may be involved in
X-linked retinitis pigmentosa, and with its rat homolog, whose expression is downregulated by v-src.27
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| Fig 6.
Expression of SRPUL mRNA in normal tissues.
Northern blot analysis of poly(A) RNA (2 µg per lane) isolated from
various human tissues, hybridized with a SRPUL cDNA probe, and
then stripped and rehybridized with a -ACTIN probe. The
mobility of a 2.5-kb RNA marker is indicated.
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| Fig 7.
Alignment of the SRPUL sushi repeat domains. The numbers
to the left of each sequence indicate the first and last residue in
each repeat, according to the SRPUL cDNA sequence deposited in Genbank
(accession no. AF060567). The conserved cysteine residues are numbered
from one to six. A consensus sequence derived from the P-selectin
(GMP-140) consensus repeat domains is also shown.18
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The predicted amino acid sequence of SRPUL also contains 30 hydrophobic
amino-terminal amino acids with features typical of a leader peptide,
including a signal cleavage site behind the serine residue at position
30.28 SRPUL lacks a hydrophobic amino acid region distal to
the leader peptide that could function as a transmembrane domain,
suggesting that it is a secreted protein. A precedent for alternative
splicing occurs in the mRNA of a related protein, P-selectin (GMP-140),
resulting in a secreted protein that lacks a transmembrane
domain.17,18 However, of six nearly full-length
SRPUL cDNAs from heart or UOC-B1(dn) cDNA libraries that we
analyzed, none corresponded to an alternatively spliced form with a
potential transmembrane domain.
Cellular localization of SRPUL-CF in FL5.12 cells.
Cellular localization of the SRPUL protein was determined by
immunofluorescence staining of FL5.12 cells transfected with a
zinc-inducible, C-terminal FLAG-tagged SRPUL cDNA clone. Sixteen hours
after the addition of zinc, these cells showed a cytoplasmic distribution of SRPUL-CF expression, suggesting localization to secretory vesicles in a pattern typical of secreted proteins
(Fig 8B and B). FL5.12 (SRPUL-CF)
cells grown without zinc showed faint cytoplasmic staining because of
low (base-line) levels of expression programmed by the pMT vector in
the absence of zinc induction (Fig 8A); mock-transfected control cells
were not stained by the antibody (Fig 8C and D).
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| Fig 8.
Subcellular localization of SRPUL-CF proteins. FL5.12
cells expressing SRPUL-CF were immunostained with an -FLAG MoAb
(M2). Simultaneous staining with DAPI permitted visualization of cell
nuclei (panels A, B, C, D). The FL5.12
cells were stably transfected with a zinc-regulated pMT vector
containing SRPUL-CF (panels A, A, B, B) or the empty
vector (panels C, C, D, D). Cells were grown in the
absence of zinc (panels A, A, C, C) or 16 hours after its
addition to the medium (100 µmol/L; panels B, B, D, D).
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Synthesis and secretion of SRPUL-CF in FL5.12 cells.
FL5.12 (SRPUL-CF) cells and FL5.12 cells transfected with empty vector
(pMT) were metabolically labeled after culture for 24 hours in the
presence and absence of zinc. SRPUL-CF was immunoprecipitated with a
murine -FLAG MoAb (M2) from cell lysates and the culture media after
continuous labeling for 3 hours. In the presence of zinc, FL5.12
(SRPUL-CF) cells produced high levels of the labeled SRPUL-CF protein
(Fig 9A). During the 3-hour incubation, a
substantial quantity of radiolabeled SRPUL-CF was recovered from the
culture medium (Fig 9B), as would be predicted for a secreted protein.
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| Fig 9.
(A) Synthesis and secretion of SRPUL-CF. Metabolically
labeled lysates of FL5.12 (SRPUL-CF)3 cells were immunoprecipitated
with the -FLAG MoAb (M2) (lanes 3 and 4) and compared with control
FL5.12 (pMT) cells (lanes 1 and 2). (B) Secretion of SRPUL-CF by FL5.12
(SRPUL-CF)3 cells (lane 4), as shown by immunoprecipitation from the
culture medium 16 hours after the addition of zinc (100 µmol/L).
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Lack of antiapoptotic activity of ANNEXIN VIII and
SRPUL-CF.
Because E2A-HLF protects IL-3-dependent murine pro-B cells from
apoptosis caused by IL-3 deprivation,10 we asked if either ANNEXIN VIII or SRPUL-CF could perform this function in the absence of
the oncoprotein. Thus, we transfected FL5.12 cells with the zinc-regulated vector encoding ANNEXIN VIII or SRPUL-CF. Independent clones were isolated after G-418 selection that expressed either of the
proteins in the presence of 100 µmol/L ZnSO4, at levels that were approximately 10-fold higher than the background level expressed by cells grown in medium lacking the metal
(Fig 10A). As shown in Fig 10B, FL5.12
cells expressing E2A-HLF survived with little loss of viability,
whereas those expressing ANNEXIN VIII or SRPUL-CF rapidly underwent
apoptosis after removal of IL-3 from the growth medium. These
observations indicate that neither protein blocks apoptosis in our test
cell system. Thus, their dysregulation by E2A-HLF in UOC-B1 human
leukemia cells probably contributes to properties of
t(17;19)+ leukemias other than prolonged cell survival.
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| Fig 10.
Effects of E2A-HLF, ANNEXIN VIII, and SRPUL-CF on the
survival of FL5.12 cells deprived of growth factor. (A) Immunoblot
analysis with antisera to HLF(C) and ANNEXIN VIII and with the -FLAG
MoAb (M2). Results are shown for three independently derived clones of
G418-resistant FL5.12 cells transfected with each of the full-length
cDNAs (FL5.12 [E2A-HLF], FL5.12 [ANNEXIN VIII] and FL5.12
[SRPUL-CF]) under control of the zinc-regulated pMT promoter as well
as controls transfected with the empty vector (FL5.12 [pMT]). (B)
Survival of FL5.12 cells expressing E2A-HLF, ANNEXIN VIII, or SRPUL-CF,
in the absence of IL-3. Cells growing exponentially in
IL-3-supplemented media for 16 hours in the presence or absence of
zinc were adjusted to 5 × 105 cells/mL on day 0, and
cultured after removal of IL-3. The cell number of viable cells is
shown for E2A-HLF-, ANNEXIN VIII-, and SRPUL-CF-expressing transfected
FL5.12 clones grown in the presence (open symbols) or absence (closed
symbols) of zinc (100 µmol/L).
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DISCUSSION |
A large number of chimeric or otherwise dysregulated transcription
factors are produced by the diverse chromosomal translocations found in
specific types of acute leukemia and sarcomas.29-31
Research into mechanisms by which these aberrant proteins subvert
normal programs of cell proliferation, differentiation, and survival is
now focused on the downstream genes transcriptionally regulated in
transformed cells. Our goal is to identify the genetic programs responsible for the transformed phenotype of ALLs with the t(17;19), which express the chimeric transcription factor E2A-HLF and carry a
poor prognosis.3,4 Two classes of genes are relevant in this regardthose mediating the antiapoptotic properties of E2A-HLF and those contributing to the unusual clinical features of patients with t(17;19)+ acute leukemias, such as coagulopathy and
hypercalcemia.
Using the RDA procedure we identified two genes, ANNEXIN VIII
and SRPUL, whose expression clearly depended on the DNA-binding activity of E2A-HLF. We also were able to show upregulation of ANNEXIN VIII in HL-60 cells by conditional expression of the
oncogenic form of E2A-HLF, providing further evidence of ANNEXIN
VIII regulation by the fusion protein. Although SRPUL mRNA
levels showed the same pattern of regulation as those at ANNEXIN
VIII in response to the dominant-negative form of E2A-HLF in UOC-B1
cells, the two genes were not coordinately upregulated in HL-60 cells.
This discrepancy likely reflects cell-type-specific differences that
determine whether individual genes are E2A-HLF responsive, such as the
requirement for other transcriptional regulatory proteins or local
epigenetic factors such as chromatin configuration or DNA methylation
status.
Our studies do not establish whether the promoter of either ANNEXIN
VIII or SRPUL is a direct target of E2A-HLF, but rather implicate these genes as elements of biochemical cascades responsive to
modulation by the chimeric transcription factor. The fact that we were
able to isolate only two differentially regulated RDA PCR products
suggests the presence of other, undetected transcripts that were
upregulated by E2A-HLF. Braun et al,12 in their RDA analysis of genes induced by the EWS-FLI fusion protein, found that at
least a 10-fold difference in transcript levels between the tester and
driver populations was needed to ensure detection of differentially
expressed mRNA species.12 Our results favor the
interpretation that RDA preferentially identifies highly regulated genes, in that both ANNEXIN VIII and SRPUL mRNA levels
declined more than 25-fold to undetectable levels as E2A-HLF
transcriptional activity was progressively suppressed through
dominant-negative interference with DNA binding.
ANNEXIN VIII belongs to a family of Ca2+-dependent
phospholipid-binding proteins that includes 13 members with similar
structures, characterized by the presence of four to eight repeats of a
70-amino acid domain and a variable region at the
N-terminus.32-34 Annexins are cytosolic proteins, and their
ability to associate with negatively charged phospholipids of the inner
membrane bilayer in the presence of Ca2+ seems to be
required for their antiphospholipase, anticoagulant, antiinflammatory,
and antiprotein kinase C activities. Annexin VIII was initially
identified as VAC-, a protein whose anticoagulant activity depends
on its phospholipid-binding properties and that also possesses
phospholipase A2 activity.24 Moreover, the
annexins drive Ca2+-dependent aggregation of secretory
granules and are postulated to have roles in endosomal vesicle
trafficking and cell-matrix interactions.32-34
Of greatest relevance to the present study, ANNEXIN VIII is
specifically upregulated in cases of acute promyelocytic leukemia (APL), but not in most other forms of chronic or acute
leukemia.16 Treatment of APL cells with
all-trans-retinoic acid induces downregulation of ANNEXIN
VIII at the transcriptional level, suggesting that it is a target
of either normal retinoic acid receptors or the PML-RAR chimeric
protein in these cells.16,17 Interestingly, patients with
acute leukemias characterized by either the E2A-HLF or PML-RAR
chimeric protein share a defining presenting featurea bleeding
diathesis that resolves after effective antileukemic therapy.3,4,35 The anticoagulant activity of ANNEXIN VIII may depend on its ability to inhibit phospholipid-dependent activation of prothrombin by factor Xa.24 The localization of ANNEXIN
VIII at the cytosolic face of the plasma membrane has been interpreted to argue against a role for this protein in the coagulopathy that affects patients with leukemias harboring PML-RAR fusion
proteins.17 However, a high rate of spontaneous leukemic
cell death is a prominent feature in newly diagnosed patients,
providing a means by which ANNEXIN VIII could be released from cells
and contribute to the bleeding problems associated with these two forms
of acute leukemia.
The ability of ANNEXIN VIII to inhibit phospholipase A2
(PLA2)24,36 would predict a contribution of the
protein to the aberrant survival of leukemic cells expressing E2A-HLF.
PLA2 activation through tumor necrosis factor (TNF)- or
Fas-mediated pathways has been linked to the production of second
messengers (such as arachidonic acid) that participate in cell death
pathways.37 Other inhibitors of cystolic PLA2,
including a trifluoromethylketone analog of arachidonic
acid,38 have been shown to partially inhibit TNF-induced
apoptosis. However, ANNEXIN VIII did not produce an antiapoptotic
effect when it was overexpressed in murine pro-B lymphocytes deprived
of growth factor (Fig 10). This suggests that any antiapoptotic
activity by ANNEXIN VIII in transformed cells would likely be
restricted to situations that involve TNF or FAS signaling, in which
PLA2 activity would be activated through specific caspase-induced cleavage.39
The SRPUL gene product possesses substantial homology with a
sushi repeat-containing protein (SRPX or ETX1), which was
independently isolated as a rat gene downregulated through the activity
of v-src.20,21,27 SRPX (ETX1) is
abundantly expressed in the retina and has been implicated as a
candidate gene for one form of X-linked retinitis pigmentosa,20,21 a heterogeneous group of human genetic
disorders that together constitute the most common cause of inherited
blindness. Although retinitis pigmentosa is characterized by the
progressive loss of retinal cells leading to retinal degeneration and
blindness, apparently because of the aberrant programmed death of
photoreceptor cells,40 there was no indication in the
present study of an antiapoptotic role for SRPUL in pro-B cells
deprived of growth factor.
The three sushi repeats (or complement control sequences) that
characterize the SRPUL protein were first identified in plasma 2 glycoprotein41 and transglutaminases, such
as factor XIIIa.26 They also occur in members of the
selectin family of adhesion molecules found on endothelium, platelets,
and leukocytes,18,19,42-44 an association with implications
for the function of SRPUL in pro-B lymphocytes transformed by E2A-HLF.
Sequence analysis of each of the three members of the selectin family
predict type-1 transmembrane proteins with N-terminal lectin-like
domains, epidermal growth factor (EGF) repeats, and variable numbers of
modules60 amino acids eachthat comprise the sushi or complement
consensus repeats.19 The lectin and EGF domains seem to
have major roles in selectin-mediated adhesion,45,46 with
the sushi repeats preventing direct contact of the lectin and EGF
regions with the cell surface, thereby enhancing adhesive
function.19 Importantly, inactivation of sushi modules in
L-selectin and E-selectin inhibits the adhesive function of both
molecules.47
The selectins are thought to contribute to the adhesive properties of
migrating tumor cells,48,49 and hence to play a role in
tumor metastasis, particularly in gastrointestinal cancers and
lymphomas.50,51 Although expression of cell adhesion
molecules is known to be upregulated in leukemic B-cell
precursors,52-56 relatively little attention has been paid
to be correlation between these observations and the dissemination of
leukemic cells or prognosis. Because E2A-HLF-positive leukemias are
characterized by bone invasion and hypercalcemia, rare observations in
early B-lineage leukemias lacking the t(17;19),4,57 we
suggest that aberrant secretion of SRPUL may contribute to this unique
feature of the disease.
We considered it likely that many of the genes stimulated directly or
indirectly by E2A-HLF do not participate in the antiapoptotic activity
of the oncoprotein, but rather contribute to the leukemic phenotype in
other ways. Thus, identifying the primary leukemogenic target of
E2A-HLF remains a crucial step in elucidating the mechanism of action
of this fusion protein. Representational difference analysis by PCR may
well lack the sensitivity to detect genes that are regulated at only
modest levels,12 so that modifications of this procedure or
perhaps completely different techniques will be needed to advance
results of the present study. One promising approach is analysis of
cDNA microarrays on glass slides,58-61 which would permit
one to identify genes whose levels are more modestly induced or
repressed as part of the complete gene program regulated by the
activity of the E2A-HLF oncoprotein. By identifying each of the genes
that are transcriptionally regulated in response to the expression of
E2A-HLF, we should ultimately be able to understand the mechanisms
through which this fusion protein transforms early lymphoid
progenitors.
| |
ACKNOWLEDGMENT |
We thank J. Wu for excellent technical assistance. We also thank F. Rauscher III for providing the pMT-CB6+ expression vector, R. Hauptmann
and G.R. Adolf for VAC--specific antiserum and MoAb, K. Ohyashiki
and K. Toyama for the HAL-01 cell line, the St Jude Tumor Processing
Laboratory for primary ALL samples with the (17;19) chromosomal
translocation, and J.& |
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Abstract
Introduction