c-myc Intron Element-binding Proteins Are Required
for 1,25-Dihydroxyvitamin D3 Regulation of
c-myc during HL-60 Cell Differentiation and the
Involvement of HOXB4*
Quintin
Pan
and
Robert U.
Simpson§
From the Department of Pharmacology, University of Michigan, School
of Medicine, Ann Arbor, Michigan 48109-0632
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ABSTRACT |
1,25-Dihydroxyvitamin D3
(1,25-(OH)2D3) suppresses c-myc
expression during differentiation of HL-60 cells along the monocytic pathway by blocking transcriptional elongation at the first exon/intron border of the c-myc gene. In the present study, the
physiological relevance of three putative regulatory protein binding
sites found within a 280-base pair region in intron 1 of the
c-myc gene was explored. HL-60 promyelocytic leukemia cells
were transiently transfected with three different c-myc
promoter constructs cloned upstream of a chloramphenicol
acetyltransferase (CAT) reporter gene. With the wild-type
c-myc promoter construct (pMPCAT), which contains MIE1,
MIE2, and MIE3 binding sites, 1,25-(OH)2D3 was able to decrease CAT activity by 45.4 ± 7.9% (mean ± S.E.,
n = 8). The ability of
1,25-(OH)2D3 to inhibit CAT activity was
significantly decreased to 18.5 ± 4.3% (59.3% reversal,
p < 0.02) when examined with a MIE1 deletion
construct (pMPCAT-MIE1). Moreover, 1,25-(OH)2D3 was completely ineffective at suppressing CAT activity in cells transfected with pMPCAT-287, a construct without MIE1, MIE2, and MIE3
binding sites (
6.5 ± 10.9%, p < 0.002).
MIE1- and MIE2-binding proteins induced by
1,25-(OH)2D3 had similar gel shift mobilities, while MIE3-binding proteins migrated differently. Furthermore, chelerythrine chloride, a selective protein kinase C (PKC) inhibitor, and a PKC
antisense oligonucleotide completely blocked the binding of nuclear proteins induced by 1,25-(OH)2D3 to
MIE1, MIE2, and MIE3. A 1,25-(OH)2D3-inducible
MIE1-binding protein was identified to be HOXB4. HOXB4 levels were
significantly increased in response to
1,25-(OH)2D3. Taken together, these results
indicate that HOXB4 is one of the nuclear phosphoproteins involved in
c-myc transcription elongation block during HL-60 cell
differentiation by 1,25-(OH)2D3.
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INTRODUCTION |
The c-myc protooncogene is linked to pathways of
cellular proliferation and differentiation. Several studies reported
that decreases in c-myc levels are required for oncogenic
cells to escape from the cell cycle and undergo terminal
differentiation. The human promyelocytic leukemia cell line, HL-60, is
an excellent model system to study mechanisms involved in cellular
differentiation. HL-60 cells can be terminally differentiated along the
monocytic/macrophage pathway by phorbol esters and
1,25-dihydroxyvitamin D3
(1,25-(OH)2D3)1
and along the granulocytic pathway by retinoic acid and
Me2SO (1-3). Accompanying this differentiation event,
c-myc levels are down-regulated by approximately 90% (2, 4,
5). Furthermore, differentiation of HL-60 cells can be triggered by
decreasing c-myc protein levels using c-myc
antisense RNA or DNA oligonucleotides (6, 7). In the presence of
1,25-(OH)2D3, transcription of c-myc
was abolished within 48 h in HL-60 cells (5). This reduction in
transcription is at least in part a result of a transcriptional elongation block at a site within the first exon/intron border of the
c-myc gene (8). Like 1,25-(OH)2D3,
phorbol esters and retinoic acid have been shown to block transcription
of the gene at the first exon/intron border (2). Several groups have
shown that 1,25-(OH)2D3 increases PKC levels
and activity (9-11). Recently, PKC
has been suggested as the
critical isozyme required for 1,25-(OH)2D3 promotion of HL-60 cell differentiation (12-15). Moreover, inhibitors of PKC activity, such as H-7 and sphinganine, blocked
1,25-(OH)2D3 promotion of cell differentiation
(10). Similarly, H-7 was able to significantly block
1,25-(OH)2D3 inhibition of c-myc
transcription. Sphinganine, a PKC inhibitor that acts on the
regulatory domain of PKC, also prevents
1,25-(OH)2D3 down-regulation of
c-myc expression (8). These results provide evidence that
PKC plays a critical role in 1,25-(OH)2D3
inhibition of c-myc transcription.
Zajac-Kaye and Levens (16, 17) identified several protein binding sites
located at intron 1 of the c-myc gene. They termed these
protein binding sites MIE1, MIE2, and MIE3. Furthermore, they reported
that mutations/deletions in MIE1, MIE2, and/or MIE3 are associated with
uncontrolled c-myc expression in numerous Burkitt's
lymphoma cell lines (18). Recently, we reported that 1,25-(OH)2D3 significantly enhances the binding
of two nuclear proteins to MIE1 in HL-60 cells (19). In the present
study, MIE1-, MIE2-, and MIE3-binding proteins were demonstrated to be required and may act in concert to regulate c-myc during
HL-60 cell differentiation. HOXB4 was identified to be the major
MIE1-binding protein. Moreover, HOXB4 protein levels were increased by
1,25-(OH)2D3 prior to monocytic differentiation
of HL-60 cells.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
HL-60 promyelocytic leukemia cells were
obtained from American Type Culture Collection and cultured in RPMI
1640 medium supplemented with 10% horse serum, 50 units/ml penicillin
G, and 50 µg/ml streptomycin. Cells were incubated at 37 °C in a
humidified atmosphere with 5% CO2. Cells used in this
study were from passage 14-45.
Plasmids--
The c-myc plasmids were generously
provided by Dr. Maria Zajac-Kaye. pMPCAT contains a 3.2-kilobase
HindIII-SstI fragment of c-myc cloned
upstream of the chloramphenicol acetyltransferase (CAT) gene (18). This
c-myc fragment includes 2328 bp upstream of exon 1, exon 1,
and the first 387 bp of intron 1, which contains the MIE1, MIE2, and
MIE3 sequences. pMPCAT-287 was constructed by deletion of
HinPI-SstI (bp 2981-3268). The MIE1 sequence was deleted from pMPCAT by polymerase chain reaction methodology using overlapping oligonucleotides to form pMPCAT-MIE1 (deletion of bp
3001-3020). A luciferase expression vector (pGL3) (Promega) was used
to normalize for transfection efficiency. The concentration and quality
of all vectors used for transfections were confirmed by agarose gel electrophoresis.
DNA Transfection and Reporter Gene Assays--
HL-60 cells
(6 × 106) were transiently transfected with 20 µg
of plasmid DNA and 2 µg of pGL3 by electroporation using a Gene Pulser II Electroporator (Bio-Rad) at 270 V and 975 microfarads. After
a 24 h recovery period, the transfected cells were divided equally
into two treatment groups. Subsequently, cells were treated with
vehicle or 100 nM 1,25-(OH)2D3 for
48 h. Cells were freeze/thawed for three cycles, and cell debris
was pelleted by microcentrifugation. Protein concentration of the
supernatants was assayed by the Bradford method (20). The amount of
cell extract and the incubation time used in the CAT assay were
optimized so that the CAT activity measured was linear. The percent of
CAT conversion was quantified on a Beckman LS8100 counter and
normalized with respect to the parent pMPCAT vector by dividing all
values by the percent conversion of the pMPCAT transfection. This
relative CAT expression value was then corrected for transfection
efficiency by dividing it by the ratio of luciferase expression between
pMPCAT-MIE1 or pMPCAT-287 and pMPCAT transfection. Luciferase activity
was measured according to the Luciferase Assay System (Promega).
Preparation of Oligonucleotides--
Oligonucleotides were
synthesized by the DNA Synthesis Core Facility at the University of
Michigan. The oligonucleotides were synthesized on Applied Biosystems
automated DNA synthesizers (Forest City, CA), employing
-cyanoethylphosphoramidite chemistry on controlled pore glass
support. MIE1 recognition sequence: 5'-AGAGTAGTTATGGTAACTGG-3'; MIE2
recognition sequence: 5'-CCTTATGAATATATTCACGC-3'; MIE3 recognition sequence: 5'-CTCCCGGCCGGTCGGACATTCCTGCTTTATTGT-3'.
Gel Shift Assay--
Nuclear protein extracts were prepared as
described previously (19). Nuclear proteins (2 µg) were incubated
with 10,000 cpm (0.1 ng) of 32P-labeled MIE1, MIE2, or MIE3
probe and 100 ng of poly[d(I-C)] in a buffer containing 20 mM HEPES (pH 7.9), 20% glycerol, 100 mM KCl,
0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl
fluoride, and 0.5 mM dithiothreitol (DTT) 30 min at
25 °C. Protein-DNA complexes were separated from free DNA probe by
electrophoresis on a high ionic strength 4% polyacrylamide gel
(acrylamide:bisacrylamide, 30:1) containing 380 mM glycine,
50 mM Tris base (pH 8.5), and 2 mM EDTA.
Thereafter, gels were dried and visualized by autoradiography.
Purification of a MIE1-binding Protein by Biotin/Streptavidin
Affinity System--
Nuclear extract (500 µg) from
1,25-(OH)2D3-treated HL-60 cells was incubated
with 20 ng of biotin-labeled MIE1 double-stranded DNA sequence in
buffer containing 20 mM HEPES (pH 7.9), 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.5 mM
phenylmethylsulfonyl fluoride, and 0.5 mM DTT for 30 min at
25 °C. Subsequently, the binding reaction mixture was incubated with
a 5 molar excess of streptavidin-agarose on a rotating wheel for 2 h. The resin was centrifuged at 14,000 rpm for 30 s, and the
supernatant (flow-through) was removed. The pellet was washed with
binding buffer two times and resuspended in elution buffer (12%
glycerol, 20 mM Tris-Cl (pH 6.8), 1 M KCl, 5 mM MgCl2, 1 mM EDTA, 1
mM DTT). After incubating on a rotating wheel for 20 min,
the resin was centrifuged at 14,000 rpm for 30 s, and the
supernatant containing the MIE1-binding proteins was collected.
Protein Sequencing--
For N-terminal protein sequencing, gels
were transferred to a polyvinylidene difluoride membrane in buffer
containing 10 mM CAPS (pH 11) for 4 h at 70 V. The
membrane was stained with Coomassie Blue, and the desired protein band
was excised. Samples were sequenced using a Proton PI 2090E Integrated
Micro-Sequencing System. Protein sequence analysis was performed by the
Protein and Carbohydrate Structure Facility at the University of Michigan.
Western Blot Analysis--
Equal amounts of nuclear protein from
each condition were run on a 10% polyacrylamide gel, and proteins were
subsequently transferred to Immobilon-P (Millipore). The membrane was
blocked with buffer containing 10 mM Tris (pH 7.4), 0.1%
Tween 20, and 2% bovine serum albumin. It was then probed for 2 h
with a HOXB4 antibody (Berkeley Antibody Co.), then washed three times
with blocking buffer and incubated for 1 h with a secondary
antibody conjugated with horseradish peroxidase (Sigma). Then, the
membrane was washed five times with Tween-TBS (10 mM Tris
and 0.2% Tween 20, pH 7.4). Finally, the membrane was developed using
enhanced chemiluminescence (NEN Life Science Products) and exposed to
x-ray film.
Statistical Analysis--
Statistical analysis was performed
using two-tailed Student's t test.
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RESULTS |
Effect of 1,25-(OH)2D3 on c-myc Promoter
Activity in Transiently Transfected HL-60 Cells--
We showed
previously that 1,25-(OH)2D3 increases the
binding of a novel 32-kDa doublet protein to the MIE1 of the
c-myc gene (19). Since the time course of this increase in
protein binding was similar to that of
1,25-(OH)2D3 inhibition of c-myc
transcription, MIE1-binding proteins were suggested to be responsible
for blocking c-myc transcription. In the present study, a
CAT reporter gene system was utilized to determine whether the
MIE-binding proteins play a functional role in
1,25-(OH)2D3 regulation of c-myc.
The wild-type c-myc construct (pMPCAT) contained 2.3 kilobases upstream of exon1, exon1, and the first 387 bp of intron 1,
which contains the MIE1, MIE2, and MIE3 binding sites. pMPCAT-MIE1 was
constructed by deleting MIE1 (bp 3001-3020) from pMPCAT. pMPCAT-287
was constructed from pMPCAT by deleting a
HinPI-SstI fragment (bp 2981-3268), which
removed the MIE1, MIE2, and MIE3 binding sites. HL-60 cells were
transiently transfected with pMPCAT, pMPCAT-MIE1, or pMPCAT-287 by
electroporation. In all experiments pGL3, a luciferase expression vector, was cotransfected to normalize for transfection efficiency. After a 24-h recovery period, transfected cells were divided equally into two treatment groups. One-half was treated with vehicle, the other
was treated with 100 nM
1,25-(OH)2D3 for 48 h. It should be noted
that these transfected cells differentiated normally in response to
1,25-(OH)2D3 as determined by nitro blue
tetrazolium dye reduction and nonspecific esterase activity (data not
shown). As shown in Fig. 1,
1,25-(OH)2D3 inhibited CAT activity by
45.4 ± 7.9% (mean ± S.E., n = 8) in cells
transfected with the wild-type c-myc construct (pMPCAT).
With the MIE1 deletion construct (pMPCAT-MIE1), 1,25-(OH)2D3 was only able to suppress CAT
activity by 18.5 ± 4.3% (mean ± S.E., n = 8); this 59.3% reversal was statistically significant
(p < 0.02). Deletion of MIE1, MIE2, and MIE3 binding sites (pMPCAT-287) completely reversed
1,25-(OH)2D3's ability to inhibit CAT activity
(
6.5 ± 10.9% inhibition by
1,25-(OH)2D3, mean ± S.E.,
p < 0.002). These transfection results indicate that MIE2- and/or MIE3-binding proteins are acting cooperatively with the
MIE1-binding proteins to block c-myc transcription.

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Fig. 1.
Effect of
1,25-(OH)2D3 on c-myc promoter
activity in transiently transfected HL-60 cells. A,
schematic representations of plasmids used in the transfection
experiments. The parent construct pMPCAT consists of a 3.2-kilobase
HindIII-SstI fragment of the human
c-myc gene cloned upstream of a CAT reporter gene.
pMPCAT-MIE1 contains a deletion of the MIE1 binding site. pMPCAT-287
contains a 287-base pair HinPI-SstI deletion and
is missing MIE1, MIE2, and MIE3 binding sites. B, CAT
activity measured in transfected HL-60 cells. Cells were transiently
transfected with pMPCAT, pMPCAT-MIE1, or pMPCAT-287 by electroporation.
Cells were cotransfected with pGL3, a luciferase expressing vector, to
control for transfection efficiency. After a 24-h recovery period,
cells were divided into two equal groups and treated with vehicle or
100 nM 1,25-(OH)2D3 for 48 h.
Values are normalized for transfection efficiency by adjusting for
luciferase activity. Data are presented as the means ± S.E. of
eight independent determinations. *, p < 0.02; **,
p < 0.002.
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Induction of MIE1-, MIE2-, and MIE3-binding Proteins by
1,25-(OH)2D3--
Since the cell transfection
data shows that MIE2 and/or MIE3 binding sites are also involved in
1,25-(OH)2D3 regulation of c-myc, we
examined if 1,25-(OH)2D3 can enhance nuclear
protein binding to MIE2 and MIE3. HL-60 cells were treated with vehicle or 100 nM 1,25-(OH)2D3 for 48 h. Nuclear proteins were isolated and assayed for MIE1, MIE2, and MIE3
binding using EMSA (Fig. 2). Consistent
with our previous study, 1,25-(OH)2D3 enhanced the binding to two nuclear proteins to the MIE1 site. MIE2-binding proteins migrated similarly to MIE1-binding proteins. This observation was not surprising, since the MIE2 binding site has sequence homology to the MIE1 binding site. Interestingly, MIE3-binding proteins induced
by 1,25-(OH)2D3 migrated differently. To
determine the specificity of these MIE-binding proteins, competition
experiments were performed (Fig. 3). MIE1
and MIE2 complexes were completely blocked by competition with
unlabeled MIE1, MIE2, and MIE3 (Fig. 3A, lanes
2-4, and Fig. 3B, lanes 7-9). Different
slower migrating MIE1 and MIE2 complexes were observed when unlabeled
MIE1 or MIE2 was used as the competitor (Fig. 3A,
lanes 2 and 3, and Fig. 3B, lanes 7 and 8). Interestingly, these slower MIE1
and MIE2 complexes were not apparent when unlabeled MIE3 was used as a
competitor, suggesting that this protein has greater affinity for MIE3
than MIE1 or MIE2. As shown in Fig. 3C, unlabeled MIE1 or
MIE2 was not as effective as unlabeled MIE3 in displacing the binding
of the MIE3 complexes. Moreover, the MIE1, MIE2, and MIE3 complexes were not displaced with a nonspecific competitor (OCT1, 100-fold excess).

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Fig. 2.
Identification of MIE1-, MIE2-, and
MIE3-binding proteins induced by
1,25-(OH)2D3. HL-60 cells were treated
with vehicle or 100 nM 1,25-(OH)2D3
for 48 h. Nuclear proteins were assayed for MIE1, MIE2, and MIE3
binding using EMSA. MIE1 sequence: 5'-AGAGTAGTTATGGTAACTGG-3';
MIE2 sequence: 5'-CCTTATGAATATATTCACGC-3'; MIE3 sequence:
5'-CTCCCGGCCGGTCGGACATTCCTGCTTTATTGT-3'. This figure is
representative of five independent experiments.
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Fig. 3.
Competition analysis between MIE1-, MIE2-,
and MIE3-binding proteins. All of the competition experiments were
performed with nuclear extracts from
1,25-(OH)2D3-treated cells. Unlabeled
MIE1, MIE2, MIE3, or OCT1 (100-fold excess) was added to the reaction
where indicated. A, 32P-MIE1; B,
32P-MIE2; C, 32P-MIE3. This figure
is representative of three independent experiments.
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Effect of a PKC Activity Inhibitor and a PKC
Antisense
Oligonucleotide on 1,25-(OH)2D3 Induction of
MIE1-, MIE2-, and MIE3-binding Proteins--
Our laboratory reported
that phorbol 12-myristate 13-acetate mimicked the effects of
1,25-(OH)2D3 by inducing a similar increase in
the binding of a 32-kDa doublet protein to MIE1 (19). Moreover, this
report showed that dephosphorylation with alkaline phosphatase completely prevented the 32-kDa proteins to bind to MIE1. However, treatment of nuclear proteins with phosphatase is a crude method to
determine the importance of protein phosphorylation. To further elucidate the role of protein phosphorylation and specifically PKC,
HL-60 cells were treated with two different protocols to inhibit
1,25-(OH)2D3 mediated increases in PKC
levels and activity. Chelerythrine chloride, a PKC activity inhibitor,
and a PKC
antisense oligonucleotide were used. Previously, our
laboratory reported the activity and specificity of the PKC
antisense used in this study (15). HL-60 cells were cotreated with 100 nM 1,25-(OH)2D3 and 3 µM chelerythrine chloride or 30 µM PKC
antisense for 48 h. Nuclear extracts were isolated and assayed for
MIE1, MIE2, and MIE3 binding using EMSA. As shown in Fig.
4A,
1,25-(OH)2D3 significantly increased the
binding of two nuclear proteins to MIE1. Extract from cells cotreated
with 1,25-(OH)2D3 and chelerythrine chloride
exhibited a mark reduction in protein binding to MIE1. Likewise, a
PKC
antisense oligonucleotide was able to significantly suppress
1,25-(OH)2D3-induced binding of proteins to
MIE1, while the PKC
sense had no effect. Similar results were
obtained using MIE2 (Fig. 4B) and MIE3 (Fig. 4C)
oligonucleotides. These observations support the notion that protein
phosphorylation via PKC
is required for
1,25-(OH)2D3 regulation of c-myc
during HL-60 cell differentiation.

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Fig. 4.
Effect of a PKC activity inhibitor and a
PKC antisense oligonucleotide on
1,25-(OH)2D3 induction of MIE1-, MIE2-, and
MIE3-binding proteins. HL-60 cells were treated with vehicle, 100 nM 1,25-(OH)2D3, 3 µM
chelerythrine chloride (CR), 100 nM
1,25-(OH)2D3 and 3 µM
chelerythrine chloride, 30 µM PKC sense ( sense), 30 µM PKC antisense ( antisense), 100 nM
1,25-(OH)2D3, and 30 µM PKC
sense or 100 nM 1,25-(OH)2D3 and 30 µM PKC antisense for 48 h. Nuclear proteins were
assayed for binding using EMSA. A, 32P-MIE1;
B, 32P-MIE2; C, 32P-MIE3.
This figure is representative of five independent experiments.
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Identification of a MIE1-binding Protein--
To purify the
MIE1-binding proteins, we utilized a biotin-streptavidin affinity
system (Fig. 5A). Nuclear
extract from 1,25-(OH)2D3-treated cells was
incubated with biotin-labeled MIE1 double-stranded oligonucleotide. The
incubation buffer, duration of incubation, and temperature were
identical to the conditions used in the EMSA. The binding reaction was
then incubated with a 5-fold molar excess of streptavidin-agarose on a
rotating wheel. The reaction mixture was centrifuged, and the
supernatant (FT) was removed. The
biotin-streptavidin-agarose pellet was washed two times (W1
and W2) with binding buffer and resuspended in elution
buffer. After incubating on a rotating wheel, the mixture was
centrifuged, and the supernatant containing the MIE1-binding proteins
was collected (E). The collected fractions were dialzyed for
2 h against 50 volumes of 20 mM HEPES (pH 7.9), 20%
glycerol, 100 mM KCl, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM
DTT. Fifty (50) µg of nuclear protein from the starting material
(SM) and the FT fraction along with the entire volume
collected from the wash 1 (W1), wash 2 (W2), and elute (E) fractions were separated on a 10% SDS-PAGE and
visualized by Coomassie blue staining. As shown in Fig. 5B,
a protein around 32 kDa was selectively retained until eluted.
Importantly, this elute fraction still maintained its capacity to bind
to MIE1 (Fig. 6, lane 4). The
retarded proteins from the elute fraction had an identical migration
pattern as compared with the starting material (Fig. 6, lane
2 versus lane 4), indicating that the
MIE1-binding proteins were still present in the elute fraction. The
sequence of the 32-kDa MIE1-binding protein (QPE(X)G) was
found to partially match (80% identity) with residues 49-53 (QPEAG)
of the human HOXB4 protein. With this information, we determined
whether the 32-kDa protein that was selectively retained by
biotin-streptavidin purification was HOXB4. When nuclear protein
extracts (1,25-(OH)2D3-treated) were subjected
to purification using MIE1-biotin, a HOXB4 antibody was able to detect
a 32-kDa protein from the starting material (SM) and elute
(E) fractions but not from the flow-through (FT) and wash (W1 and W2) fractions (Fig.
6B). To further support the identification of the 32-kDa
protein as HOXB4, supershift experiments were performed using a HOXB4
antibody (Fig. 7). Nuclear protein extracts used in the supershift experiments were subjected to biotin-streptavidin purification. Consistent with Fig. 6, MIE1 binding
was still observed with the elute fraction from
1,25-(OH)2D3-treated nuclear proteins. A HOXB4
antibody was able to completely block the binding of nuclear proteins
to MIE1. The nonimmune serum had no effect. These results demonstrate
that the 32-kDa HOXB4 protein is the major MIE1-binding protein.

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Fig. 5.
Purification of a MIE1-binding protein by
biotin-streptavidin affinity system. A, purification
scheme for a MIE1-binding protein. Nuclear extract (500 µg) from
1,25-(OH)2D3 (100 nM for 48 h)-treated cells was incubated with biotin-labeled MIE1 double-stranded
oligonucleotide in binding buffer for 30 min at 25 °C. Subsequently,
the binding reaction was incubated with a 5-fold molar excess of
streptavidin-agarose on a rotating wheel for 2 h. The reaction
mixture was centrifuged at 14,000 rpm for 30 s, and the
supernatant (FT) is removed. The pellet was washed two times
with binding buffer (W1 and W2) and resuspended
in elution buffer. After incubating on a rotating wheel for 20 min, the
mixture was centrifuged at 14,000 rpm for 30 s, and the
supernatant containing the MIE1-binding proteins was collected
(E). B, SDS-PAGE analysis of fractions
collected from the MIE1 biotin-streptavidin affinity system. Fifty (50)
µg of nuclear protein from the starting material (SM) and
the FT fraction along with the entire volume collected from
the W1, W2, and E fractions were
separated on a 10% SDS-PAGE and stained with Coomassie Blue.
First lane: STD, protein standard; second
lane: SM, starting material; third lane:
FT, flow-through; fourth lane: W1,
wash 1; fifth lane: W2, wash 2; sixth
lane: E, elute. This figure is representative of three
independent experiments.
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Fig. 6.
Analysis of elute fraction collected from
MIE1 biotin-streptavidin affinity system. A, EMSA
analysis using 32P-MIE1. HL-60 cells were treated with
vehicle or 100 nM 1,25-(OH)2D3 for
48 h. Nuclear proteins were extracted and designated as the
starting material (SM). Half of the SM was further subjected
to biotin-streptavidin purification and the elute (E)
fraction was collected. MIE1 binding was determined with the
SM and E fractions from vehicle and
1,25-(OH)2D3-treated cells.
B, Western blot analysis for HOXB4. Nuclear
extract from 1,25-(OH)2D3-treated cells were
subjected to biotin-streptavidin purification. The fractions collected
were separated on a 10% SDS-PAGE. SM, starting material;
FT, flow through; W1, wash 1; W2, wash
2; E, elute. This figure is representative of three
experiments.
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Fig. 7.
Supershift analysis of
1,25-(OH)2D3-inducible MIE1-binding
proteins. Elute fraction collected from biotin-streptavidin
purification were preincubated for 20 min in the absence of antibody
( ) or in the presence of either nonimmune or HOXB4 specific antibody.
The reaction mixture was then incubated with 32P-MIE1 for
30 min at 25 °C and analyzed by EMSA. First lane, elute
fraction from vehicle-treated cells; second lane, elute
fraction from 1,25-(OH)2D3-treated cells;
third lane, elute fraction from
1,25-(OH)2D3-treated cells and HOXB4 antibody;
fourth lane, elute fraction from
1,25-(OH)2D3-treated cells and nonimmune
antibody. This figure is representative of five independent
experiments.
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Effect of 1,25-(OH)2D3 on HOXB4 Protein
Levels--
HL-60 cells were treated with vehicle or 100 nM 1,25-(OH)2D3 for 48 h.
HOXB4 protein levels were determined by Western blot analysis (Fig.
8). HOXB4 has been characterized as a
phosphoprotein with a molecular mass of 33 kDa (21). HOXB4 levels were
significantly increased in cells treated with
1,25-(OH)2D3 by 125 ± 15%
(n = 5). Interestingly,
1,25-(OH)2D3 also increased the levels of a 60-kDa protein that is recognized by the HOXB4 antibody by 95 ± 12% (n = 5). We do not know the relationship between
the 60-kDa protein and the 32-kDa HOXB4.

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Fig. 8.
Effect of
1,25-(OH)2D3 on HOXB4 protein levels.
HL-60 cells were treated with vehicle or 100 nM
1,25-(OH)2D3 for 48 h. HOXB4 protein
levels were determined by Western blot analysis and quantified by
densitometry. This figure is representative of five independent
experiments.
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DISCUSSION |
The c-myc protooncogene has been shown to be important
in the control of cellular proliferation and differentiation.
Overexpressing c-myc in transgenic mice results in high
incidences of widespread neoplasia (22, 23). Conversely, transgenic
mice carrying the null mutations for c-myc die in
utero between 9.5 and 10.5 days (24). Moreover, c-myc
levels are elevated in both experimentally induced and naturally
occurring tumors (25). Recently, c-myc has been reported to
play a role in the regulation of telomerase activity. Several groups
have shown that differentiation of HL-60 cells with retinoic acid or
phorbol 12-myristate 13-acetate results in a significant reduction of
telomerase activity (26-28). Fujimoto and Takahashi (29) reported that
a c-myc antisense oligonucleotide inhibits telomerase
activity in three leukemia cell lines, HL-60, U937, and K562.
Telomerase is a ribonucleoprotein complex that adds telomeric repeats
onto the ends of chromosomes during the replicative phase of the cell
cycle. Without telomerase activity cells will withdraw from the cell
cycle and undergo cell death. Telomerase activity has been detected in
a wide variety of human tumors, but only in a few normal somatic cells
(30). Thus, differentiation of tumor cells is associated with decreased
c-myc expression and telomerase activity.
Regulation of c-myc gene expression is complex and has been
shown to involve several mechanisms, including changes in transcription initiation, transcription elongation, RNA stability, and RNA
translation (4, 31). Previously, we reported that binding of a 32-kDa doublet protein to MIE1 may be a mechanism used by
1,25-(OH)2D3 to regulate c-myc
transcription during HL-60 cell differentiation (19). The results from
our cell transfection data indicate that MIE1-, MIE2-, and MIE3-binding
proteins are required and may cooperate to down-regulate
c-myc during 1,25-(OH)2D3 promotion
of HL-60 cells (Fig. 1). In addition, we demonstrated that
1,25-(OH)2D3 induces nuclear proteins to bind
to MIE2 and MIE3. As shown in Fig. 3, a 100-fold excess of unlabeled
OCT1 was unable to displace the binding of specific nuclear proteins to
MIE1, MIE2, and MIE3. Unlabeled MIE1 or MIE2 was able to completely
displace the binding of two retarded bands to 32P-MIE1 and
caused the appearance of a higher molecular weight MIE1 complex. This
higher molecular weight MIE1 complex was visible in the absence of
unlabeled MIE1 or MIE2 if the film was allowed to expose for a longer
period of time (data not shown). These observations suggest that the
higher molecular weight binding protein has a lower affinity for MIE1.
The same results were obtained using 32P-MIE2, suggesting
that the same proteins bind to MIE1 and MIE2. Moreover, unlabeled MIE3
was able to block the binding of nuclear proteins to MIE1 and MIE2
(Fig. 3A, lane 4). Unlabeled MIE3 was more
effective at displacing nuclear proteins to bind to
32P-MIE3 than unlabeled MIE1 or MIE2 (Fig. 3C,
lanes 12-14). It should be noted that the fastest migrating
MIE3 band appears to bind with specificity to MIE3. Taken together,
these results suggest that these
1,25-(OH)2D3-inducible proteins are capable of
binding to MIE1, MIE2, or MIE3 with different specificities and affinities.
We and others (9-11) showed previously that
1,25-(OH)2D3 promotes the differentiation of
HL-60 cells by increasing PKC levels. In addition, it was demonstrated
that 1,25-(OH)2D3 must also provide the
cofactors, calcium and phospholipids, to chronically activate PKC in
order to promote HL-60 cell differentiation (32). Recently, we reported
that PKC
antisense oligonucleotides were able to block
1,25-(OH)2D3-mediated increases in PKC
levels as well as 1,25-(OH)2D3 promotion of
HL-60 cell differentiation (15). Using the same PKC
antisense
construct, MIE1, MIE2, and MIE3 complexes induced by
1,25-(OH)2D3 were completely abrogated (Fig.
4). Similarly, chelerythrine chloride, a selective PKC activity
inhibitor, was able to significantly reduce the binding of MIE1, MIE2,
and MIE3 to the nuclear proteins. These data suggest that
c-myc regulatory proteins must be phosphorylated via PKC
in order to bind to their respective MIE sites.
The results from the biotin-streptavidin purification showed that a
32-kDa protein was retained by biotin-MIE1. Previously, we reported
that the predominant MIE1-binding protein is a 32-kDa doublet protein
as determined by SouthWestern blot analysis. From this, we were
interested in identifying the 32-kDa protein isolated from the
biotin-streptavidin purification. The amino acid sequences obtained for
the 32-kDa protein have similarities to the human HOXB4 protein. Also,
the characteristics of HOXB4 was similar to our unknown 32-kDa
MIE1-binding protein. HOXB4 and the 32-kDa MIE1-binding protein are
phosphoproteins with similar molecular weights. Additionally, both
proteins bind DNA and act as transcriptional regulators. To determine
whether the 32-kDa MIE1-binding protein was HOXB4, a supershift EMSA
was performed using a HOXB4 antibody. As shown in Fig. 7, a HOXB4
antibody was able to block the binding of nuclear proteins to MIE1.
Most likely, the HOXB4 antibody is interacting with the MIE1 binding
site on HOXB4 since addition of 32P-MIE1 prior to antibody
incubation partially reverses the block in complex formation (data not
shown). Moreover, a HOXB4 antibody immunodetected the 32-kDa protein
that was affinity purified using biotin-MIE1 (Fig. 6). Taken together,
our results strongly support the identification of the 32-kDa
MIE1-binding protein as HOXB4.
The homeobox (HOX) genes are a family of transcriptional regulators
that contain a conserved 183-nucleotide sequence. This nucleotide
sequence encodes a 61-amino acid domain, the homeodomain, and includes
a helix-turn-helix DNA binding motif (32, 33). Several investigators
have examined the role of individual homeobox genes in cell lines and
in primary hematopoietic tissues (34-36). From these studies, evidence
exists to suggest the possibility of a correlation between expression
of individual HOX genes and hematopoietic cell phenotype (36, 37).
Studies utilizing human leukemia cells have demonstrated that cells
representing various stages of hematopoietic differentiation exhibit
different patterns of HOX gene expression (38). Furthermore, HOX genes
have been involved in translocation events in certain leukemia cells,
suggesting that mutant/nonfunctional forms of these proteins may be
important in oncogenesis (39-41). Studies have shown that HOX proteins
are able to bind to regulatory sites to control gene expression. HOX3C, HOX4C, HOX4D, and HOX4E are able to transactivate the HOX3D promoter in
both HeLa and NIH3T3 cells (42). In addition, Zappavigna et
al. (43) reported that HOX4C and HOX4D are able to transactivate the HOX4D promoter, whereas HOX4E is not. Relevant to our finding, Vallerga et al. (44) demonstrated that the HOX 2.2 protein
is able to bind to the second intron and the 3' enhancer region of the
-globin gene, important regulatory sites for globin expression. Our
studies indicate that HOXB4 is the major MIE1-binding protein. Thus, it
is possible that HOXB4 is able to block transcription elongation and
thereby negatively regulate c-myc expression during cell
differentiation. Alternatively, it is possible that HOXB4 is
heterodimerizing with a distinct MIE1-binding protein to form a
cooperative MIE1 binding complex. Several studies have shown that HOX
proteins heterodimerize with non-HOX proteins to form DNA binding
complexes (45, 46). Shen et al. (46) reported that the
Meis1-HOX heterodimer binding complex dissociates at a much slower rate
from its target DNA than Meis1 alone. Our data does not exclude any of
these possibilities but indicates that HOXB4 is involved in the complex
regulation of c-myc expression.
Several investigators have reported that modulation of HOX expression
can affect myeloid differentiation. Overexpression of HOXB8 in a murine
myeloid cell line inhibited the ability of interleukin-6 to promote
differentiation (47). Lill et al. (48) demonstrated that
overexpression of HOXB7 in HL-60 cells blocked granulocytic differentiation in response to retinoic acid or Me2SO, but
did not potentiate monocytic differentiation promoted by
1,25-(OH)2D3. Moreover, they reported that
overexpression of HOXB7, by itself, is not sufficient to promote
monocytic differentiation, suggesting that other HOX proteins must be
involved in this differentiative process. Recently,
1,25-(OH)2D3 has been shown to increase HOXB7 expression in HL-60 cells prior to monocytic differentiation (48). In
the present study, HOXB4 protein levels in HL-60 cells were significantly increased upon exposure to
1,25-(OH)2D3. Interestingly, in differentiated
HL-60 cells (1,25-(OH)2D3 for 72 h), HOXB4
protein levels were similar to untreated cells (data not shown). This observation suggests that HOXB4 is involved in the process of initiating differentiation but not in maintaining a mature cell phenotype.
In summary, these results demonstrate that MIE1, MIE2, and MIE3 binding
sites are necessary for 1,25-(OH)2D3
down-regulation of c-myc. Also, nuclear proteins binding to
MIE1, MIE2, and MIE3 are blocked by inhibition of PKC activity and
decreased PKC
levels. This study further reveals that a major
MIE1-binding protein is HOXB4. Moreover, HOXB4 protein levels are
regulated by 1,25-(OH)2D3 during HL-60 cell differentiation.
 |
ACKNOWLEDGEMENT |
We thank Dr. Maria Zajac-Kaye for providing
the c-myc promoter plasmids.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants DE10337 and CA69568.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.
Newton-Loeb Fund/The University of Michigan Cancer Center
Predoctoral Fellow.
§
To whom correspondence should be addressed. Tel.: 734-763-3255;
Fax: 734-763-4450; E-mail: robsim{at}umich.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
1, 25-(OH)2D3, 1,25-dihydroxyvitamin
D3;
PKC, protein kinase C;
MIE, c-myc intron
element;
CAT, chloramphenicol acetyltransferase;
EMSA, electrophoretic
mobility shift assay;
CAPS, 3-(cyclohexylamino)-1-propanesulfonic acid;
DTT, dithiothreitol;
bp, base pair(s);
PAGE, polyacrylamide gel
electrophoresis.
 |
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