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Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3391-3400
Characterization of HOX Gene Expression During Myelopoiesis: Role of
HOX A5 in Lineage Commitment and Maturation
By
John F. Fuller,
Jeanne McAdara,
Yifah Yaron,
Mark Sakaguchi,
John
K. Fraser, and
Judith C. Gasson
From the Department of Biological Chemistry, UCLA School of Medicine,
the Division of Hematology-Oncology, Department of Medicine, the
Jonsson Comprehensive Cancer Center, and the Molecular Biology
Institute, UCLA, Los Angeles, CA.
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ABSTRACT |
During the process of normal hematopoiesis, proliferation is tightly
linked to maturation. The molecular mechanisms that lead to production
of mature effector cells with a variety of phenotypes and functions
from a single multipotent progenitor are only beginning to be
elucidated. It is important to determine how these maturation events
are regulated at the molecular level, because this will provide
significant insights into the process of normal hematopoiesis as well
as leukemogenesis. Transcription factors containing the highly
conserved homeobox motif show considerable promise as potential regulators of hematopoietic maturation events. In this study, we
focused on identification and characterization of homeobox genes of the
HOX family that are important in regulating normal human myeloid
differentiation induced by the hematopoietic growth factor,
granulocyte-macrophage colony-stimulating factor (GM-CSF). We have
identified three homeobox genes, HOX A5, HOX B6, and HOX B7, which are
expressed during early myelopoiesis. Treating bone marrow cells with
antisense oligodeoxynucleotides to HOX A5 resulted in inhibition of
granulocytic/monocytic hematopoiesis and increased the generation of
erythroid progenitors. Also, overexpression of HOX A5 inhibited
erythroid differentiation of the K562 cell line. Based on these
observations, we propose that HOX A5 functions as an important
regulator of hematopoietic lineage determination and maturation.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HOMEOBOX GENES encode proteins containing
a conserved DNA binding domain of 61 amino acids that forms a
helix-turn-helix motif, referred to as the homeodomain. Originally
described in Drosophila, the homeobox genes are important
regulators of embryonic development, with their spatio-temporal
patterns of expression controlling the positioning of body axes,
pattern formation, and the expression of many structural genes. In
humans, the majority of homeobox genes are arranged in four clusters on
chromosomes 2, 7, 12, and 17. These so-called class-1 human homeobox
genes are highly homologous to the Antennapedia class of
Drosophila homeobox genes and are referred to as "HOX"
genes (reviewed in Akam1 and De Robertis et
al2).
Despite an increasing body of knowledge regarding hematopoietic growth
factors and their receptors, details of the nuclear effectors
associated with proliferative and differentiative signals are not yet
clear. Members of the homeobox gene family show considerable promise as
candidates for genes involved in the regulation of hematopoietic
maturation events (reviewed in Lawrence and Largman3). Recent investigations have revealed differential, lineage-restricted patterns of HOX gene expression in leukemic cell lines.3,4 A regulatory role for HOX genes in blood cell maturation can be inferred from experiments in which expression of a specific HOX gene
has been blocked or amplified in leukemic cell lines. In such cases,
the cell's normal maturation pathway is subverted.5-9 Moreover, abnormal homeobox gene expression has been described in a
number of leukemias.3,10
The study of homeobox genes in leukemic cells provides convincing
evidence that aberrant expression can play an important role during
leukemogenesis. However, it is difficult to ascertain the normal
function of these genes in hematopoiesis, because unregulated homeobox
gene expression has been identified as playing a role in the
transformed phenotype.3,10 Therefore, we have designed a
reverse transcriptase-polymerase chain reaction (RT-PCR)-based strategy to identify HOX genes that are expressed during
granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced
differentiation of normal human bone marrow cells. GM-CSF stimulates
the proliferation and maturation of multipotential progenitors into
cells of predominantely granulocytic and monocytic/macrophage lineages.
We characterized the function of one of these genes, HOX A5, using an
antisense oligodeoxynucleotide (ODN) strategy to suppress its
expression in both colony assays and an ex vivo expansion regimen and
by enforcing its expression in the K562 cell line. Our results suggest an important role for HOX A5 in the regulation of myeloid cell differentiation.
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MATERIALS AND METHODS |
RT-PCR screen for HOX gene expression in GM-CSF-stimulated primary
bone marrow cells.
Human bone marrow was obtained from a normal donor following informed
consent. Mononuclear cells were purified by gradient centrifugation on
Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) for 1 hour at
400g. The resulting buffy coat was removed and washed twice
with phosphate-buffered saline (PBS). Cells were then suspended in
0.9% methylcellulose containing Iscove's modified Dulbecco's media
(IMDM) supplemented with 30% fetal calf serum (FCS) and 1 nmol/L human
GM-CSF (generously provided by Dr Larry Souza, Amgen, Thousand Oaks,
CA) and incubated at 37°C, 5% CO2, 100% humidity.
After 4 days, GM-CSF-induced clusters containing 4 to 8 cells were
picked, pooled, and washed twice in PBS. Approximately 5 × 103 cells were collected. mRNA was isolated from the pooled
clusters using the QuickPrep Micro mRNA Purification Kit (Pharmacia)
and resuspended in 16 µL of H2O. This mRNA (4 µL) was
reverse transcribed into cDNA using AMV reverse transcriptase
(Boehringer Mannheim, Indianapolis, IN) in a 20-µL
reaction volume, per the manufacturer's instructions.
This cDNA was used in a PCR reaction using degenerate primers
complementary to conserved sequences belonging to the mammalian Antennapedia class of HOX genes: 5' primer,
5'-AT(A/C/T)TA(C/T)CC(A/C/G/T)TGGATG-3'; and 3'
primer,
5'-TTTCAT(A/G)C(T/G)(A/G)C(G/T)(A/G)TT(C/T)TG(G/A)AACCA-3'. Oligonucleotide primers were synthesized on a PCR Mate oligonucleotide synthesizer (Applied Biosystems, Foster City, CA) and purified by
affinity chromatography using a Nensorb column per the manufacturer's instructions (DuPont, Boston, MA). To avoid the presence
of false-positive PCR products resulting from genomic DNA contamination
in the mRNA, this primer set spans an intron, and parallel cDNA
reactions lacking reverse transcriptase were also performed as a control.
The PCR reaction mix contained 5 µL cDNA, 0.5 mmol/L of each 3'
and 5' primer, 0.25 mmol/L of each dNTP (Boehringer Mannheim), 16.6 mmol/L (NH4)2SO4, 67 mmol/L
Tris-HCl (pH 8.8; Sigma, St Louis, MO), 6.7 mmol/L
MgCl2 (Sigma), 6.7 mmol/L EDTA (Sigma), 0.17 mg/mL bovine
serum albumin (BSA; Sigma), 10% dimethyl sulfoxide (DMSO; Sigma), and
1.25 U of Taq polymerase (Boehringer Mannheim). PCR cycling was
performed in a Perkin-Elmer Cetus (Norwalk, CT)
thermocycler as follows: 94°C for 1 minute, 45°C for 1 minute, and 72°C for 2 minutes for 35 cycles, followed by a
72°C extension for 10 minutes. Five microliters of this reaction
was then reamplified in a fresh reaction mix under the same conditions
for 25 cycles. This reamplified PCR product was then directly ligated
into the TA vector (Invitrogen, Carlsbad, CA). Clones were
sequenced to identify the numbers and types of homeobox genes present.
Complete sequences were identified with the GENBANK database (Bethesda, MD).
Analysis of HOX A5 expression in CD34+ cells.
Bone marrow was obtained from a normal, healthy volunteer after
informed consent was received. CD34+ cells were purified
using the Miltenyi Mini Macs CD34 antibody and column kit (Miltenyi
Corp, Sunnyvale, CA). A portion of these CD34+
cells was plated into colony assays containing IMDM, 0.3% agar, 20%
FCS, 50 ng/mL each of interleukin-3 (IL-3), IL-6, stem cell factor
(SCF), and granulocyte colony-stimulating factor (G-CSF) and 2 U
erythropoietin (Epo). CD34+ cells (5 × 105) were plated into 2 mL of IMDM + 20% fetal bovine
serum (FBS) with or without 1 nmol/L GM-CSF. After 72 hours, cells were
harvested, and a portion of each population was plated into colony
assays containing G-CSF, SCF, IL-3, IL-6, GM-CSF, and Epo. From 5 × 105 cells of each population, mRNA was extracted
using the QuickPrep Micro mRNA Purification Kit (Pharmacia). mRNA
samples were analyzed by HOX A5 semiquantitative RT-PCR.
HOX A5-specific semiquantitative RT-PCR.
HOX A5-specific primers (5' primer,
5'-CGCCGGCAGCACCCACATCAG-3'; 3' primer,
5'-TTCCGGGCCGCCTATGTTGT-3') and glyceraldehyde phosphate
dehydrogenase (GAPDH)-specific primers (5' primer,
5'-TGATGACATCAAGAAGGTGGTGAAG-3'; 3' primer,
5'-TCCTTGGAGGCCATGTGGGCCAT-3')11 were
synthesized and purified over a Nensorb column, as described above.
mRNA was treated with RNase-free DNase in a buffer containing 10 mmol/L Tris-HCl, 1 mmol/L dithiothreitol (DTT), 10 mmol/L
MgCl2, 0.025 U/µL RNase inhibitor (Boehringer Mannheim),
and 5 ng/µL RNase-free DNase (Boehringer Mannheim) for 15 minutes at
37°C. DNase was inactivated and removed from the samples by heating
to 70°C for 10 minutes, followed by the addition of EDTA to 10 mmol/L, phenol-chloroform extraction, and ethanol precipitation.
Reverse transcription of equal amounts of mRNA was performed using
SuperScript II reverse transcriptase and an oligo(dT) primer according
to the manufacturer's instructions (GIBCO-BRL, Gaithersburg,
MD). Controls for DNA contamination consisted of the
entire reverse transcription mix minus the reverse transcriptase and
were performed in parallel. cDNA (5 µL) was added to a 50-µL PCR
reaction mix containing 0.8 µmol/L HOX A5 or GAPDH primer pairs, 3.75 U Taq polymerase (GIBCO), 25 µmol/L of each deoxynucleotide, and 1.5 mmol/L MgCl2 in the manufacturer's PCR buffer. HOX A5 PCR
was performed in a thermocycler (Perkin-Elmer Cetus) under the
following conditions: 94°C for 1 minute, 60°C for 1 minute, and
72°C for 1.5 minutes for 45 cycles, followed by a 72°C
extension for 10 minutes. GAPDH PCR was performed in parallel
as follows: 94°C for 2 minutes, 55°C for 1.5 minutes, and 72°C for 1.5 minutes for 35 cycles, followed by a 72°C
extension for 10 minutes. PCR products were separated by
electrophoresis on a 3% NuSieve Agarose-TBE gel (FMC BioProducts,
Rockland, ME). Gels were stained with ethidium bromide,
and UV-visible bands were digitally recorded using a Speedlight
Platinum gel documentation system (Lightools Research, Encinitas,
CA) and quantitated using ImageQuant v1.1 software
(Molecular Dynamics, Sunnyvale, CA). The results of
titration experiments show parallel linear increases in signal with
input concentrations of mRNA in the range of 0.1 to 5 µg. HOX A5
sequences were identified by a Southern analysis probe using a
random-primed radiolabeled fragment of the first 485 nucleotides of HOX
A5 cDNA, excluding the homeodomain. Southern blots were developed on a
Molecular Dynamics PhosphorImager 445SI.
Cloning of HOX A5.
PCR primers specific for the HOX A5 sequence (5' primer,
5'-AATGAGCTCTTATTTTGTAAACTC-3', and 3' primer,
5'-TCAGATACTCAGGGACGGAAGGC-3') were synthesized and
purified as described above. mRNA was extracted from the myelomonocytic
cell line, U937, by the Pharmacia Micro QuickPrep kit and cDNA made
using AMV reverse transcriptase (Boehringer Mannheim) per the
manufacturer's instructions. U937 cDNA (5 µL) was amplified in a PCR
mix containing 2.5 U Taq polymerase (Boehringer Mannheim), 1 µmol/L
of each HOX A5-specific primer, 200 µmol/L of each nucleotide, 1 mmol/L MgCl2, and 0.5% DMSO in the manufacturer's supplied buffer. Cycling parameters are as follows: 94°C for
1.5 minutes, 50°C for 1.5 minutes, and 72°C for 1.5 minutes for 35 cycles, followed by a 72°C extension for 10 minutes.
PCR products were subcloned into the TA vector as described above, and
dideoxynucleotide sequencing was performed to confirm identity with
published sequence.12 A putative isoform of HOX A5 was also
isolated using this strategy. This cDNA contains a 4-nucleotide
deletion at position 770, resulting in a protein truncated at the
3' end of the homeodomain. No difference is observed when the
full-length and truncated HOX A5 clones are compared in side-by-side
biological assays.
Construction of K562-LA5SN-expressing cells.
HOX A5 cDNA was subcloned into the retroviral vector, LXSN (generously
provided by Dusty Miller, Fred Hutchinson Cancer Research Center,
Seattle, WA), to create the construct LA5SN. LA5SN was transfected
using lipofectin into the ecotropic murine fibroblast packaging line,
GPE. Culture supernatants from LA5SN-containing GPE cells were
collected and used to infect the amphotropic murine fibroblast
packaging line, PA317. K562 cells were transduced with LA5SN by
cocultivation with irradiated PA317 packaging cells expressing LA5SN.
Antisense oligonucleotide preparation.
HOX A5 antisense (5'-TACAAAATAAGAGCTCAT-3'), HOX A5
sense (5'-ATGAGCTCTTATTTTGTA-3'), and HOX A5 nonsense
(scrambled HOX A5 antisense: 5'-CACAACATAAGTAATTAG-3') ODN
were synthesized as described above, purified by butanol extraction and
ethanol precipitation, and resuspended in serum-free hematopoietic stem
cell medium (GIBCO-BRL). The antisense ODN was designed to
recognize the initial 18 nucleotides of the HOX A5 mRNA, beginning with
the translational start codon. The specificity of the antisense and
sense oligodeoxynucleotides for HOX A5 sequences and the nonspecificity
of the nonsense oligodeoxynucleotide for any known sequence was
determined by screening GENBANK.
Assay of antisense ODN activity.
K562-LA5SN cells (107) were plated in 3 mL of X-Vivo-10
serum-free medium (BioWhittaker, Walkersville, MD)
containing 15 µmol/L HOX A5 antisense, sense, or nonsense ODN for 24 hours. Control cultures containing no oligodeoxynucleotide were also
prepared. Total RNA was extracted from treated cells using a urea
lysis/sodium dodecyl sulfate (SDS) method. RNA was processed as
described above for the semiquantitative PCR protocol and subjected to
the same RT-PCR procedure, with the following modifications: 2 µL of
cDNA and 2.5U of Taq polymerase were used per 50-µL PCR reaction, HOX A5 PCR was shortened to 35 cycles, and GAPDH PCR was shortened to 25 cycles. Quantitation of PCR products was performed using the Speedlight
Platinum gel documentation system and ImageQuant v1.1 software, as
described above.
Antisense HOX A5 ODN colony-forming unit-granulocyte-macrophage
(CFU-GM) colony assay.
Human bone marrow was obtained from a normal donor after informed
consent was received. Mononuclear cells were purified by gradient
centrifugation on Ficoll-Hypaque (Pharmacia) for 40 minutes at
400g. The resulting buffy coat was removed and washed twice with PBS. Cells were preincubated overnight in indicated amounts of ODN
in serum-free medium (HSCM; GIBCO-BRL). Colony assays were performed by
plating 105 viable cells in HSCM containing 0.3% agar
(DIFCO, Detroit, MI), 1 nmol/L GM-CSF, and antisense,
sense, or nonsense ODN added at indicated concentrations in a total
volume of 0.5 mL in triplicate into the wells of a 24-well plate.
Colonies were incubated at 37°C, 5% CO2, 100%
humidity for 14 days. CFU-GM were scored in a blinded fashion at day 14 when more than 50 cells were present.
Antisense HOX A5 ODN ex vivo expansion assay.
Ex vivo expansion assays were performed using a modification of our
previously published procedure.13 Human bone marrow mononuclear cells from a normal donor were purified as described above.
CD34+ cells were separated from the mononuclear cells using
a Ceprate LC column per the manufacturer's instructions (Cellpro,
Bothell, WA). A portion of these CD34+ cells
was plated into colony assays containing IMDM; 0.3% agar; 20% FCS; 50 ng/mL each of IL-3, IL-6, SCF, and G-CSF; and 2 U of Epo. The remaining
CD34+ cells (1.5 × 104) were incubated in
0.5 mL HSCM containing 50 ng each of IL-3, IL-6, SCF, and G-CSF.
Antisense, sense, or nonsense ODN to HOX A5 were added to a
concentration of 15 µmol/L. Cells were incubated at 37°C, 5%
CO2, 100% humidity for 10 days, and cultures were resupplemented with fresh medium, growth factors, and ODN after 5 days.
On day 10, the cells were washed twice in PBS and plated in triplicate
into 0.5-mL colony assays, with each well containing 5 × 103 cells in IMDM; 0.3% agar; 20% FCS; 50 ng each of
IL-3, IL-6, SCF, and G-CSF; and 2 U Epo. Colony assays were incubated
at 37°C, 5% CO2, 100% humidity for 14 days. CFU-GM
and burst-forming units-erythroid (BFU-E) were scored in a blinded
fashion on day 14, based on colony morphology and color.
Construction of FLAG-tagged HOX A5-Thy 1.2 expression vector.
A FLAG epitope adapter was created by synthesizing coding
(5'-AAAGGATCCATGGACTACAAAGACGATGACGATAAAGAATTCATA-3';
FLAG-encoding sequence underlined) and complementary
(5'-TATGAATTCTTTATCGTCATCGTCTTTGTAGTCCATGGATCCTTT-3') single-stranded DNA on a PCR Mate oligonucleotide synthesizer (Applied
Biosystems). These oligonucleotides were purified by affinity
chromatography using a Nensorb column per the manufacturer's instructions (DuPont) and annealed by heating to 95°C for 5 minutes, followed by slow cooling to room temperature. This adapter
contains a 5' BamHI and 3' EcoRI sites for
cloning purposes and an ATG translational start site. The FLAG epitope
adapter was ligated to the 5' end of the HOX A5 cDNA, and the
resultant FLAG-HOX A5 construct was subcloned into the BSVprXThy
vector,14 a derivative of the pCMV-thy-1
vector,15 both kindly provided by the laboratory of Dr
Irvin Chen (UCLA, Los Angeles, CA). The BSVprXThy vector contains two
cytomegalovirus (CMV) promoters, one driving expression of the cell
surface antigen, murine Thy1.2, and the other driving expression of the
HA nonapeptide and the HIV-1 vpr gene. The HA nonapeptide and
the vpr gene were removed by HindIII/Xba I
digestion and replaced by the FLAG-HOX A5 construct. Here, we refer to
this FLAG-tagged HOX A5/Thy 1.2 coexpression vector as pFAT (for
FLAG-A 5-Thy-1). The pCMV-thy-1 vector expressing only murine
Thy 1.2 was used as a control.
Transient transfection and sorting of K562 cells with FLAG-tagged
HOX A5.
K562 cells (107) were electroporated with 14 nmol/L pFAT or
pCMV-thy-1 expression plasmids. Electroporations were performed in
1.2× RPMI-1640 containing 20% FBS at 250 V using a Bio-Rad GenePulser (Bio-Rad, Hercules, CA). Electroporated cells
were placed in IMDM + 10% FBS. After 24 hours, electroporated cells were washed in PBS and replated in fresh medium. After electroporation (24 hours), cells were harvested for sorting. Sorting of Thy
1.2-positive cells was performed using the Miltenyi MiniMacs column
system, employing magnetic beads conjugated to an antimouse Thy 1.2 antibody supplied with the kit. Purification was performed according to the manufacturer's directions, with the exception that the purified Thy 1.2-positive eluate was run over a second MiniMacs column to
further enrich the population for high-expression cells. Analysis of
flow-through and eluate fractions from the Miltenyi columns for Thy
1.2-positive cell purity and recovery was performed by fluorescence-activated cell sorting (FACS; Becton Dickinson, Franklin Lakes, NJ) using antimouse Thy 1.2 (Caltag, Burlingame,
CA) antibody. Expression of FLAG-tagged HOX A5 in column
fractions was analyzed by Western blot using an anti-FLAG epitope
antibody (Eastman Kodak, Rochester, NY). For the Western
blots, 105 cells were resuspended in SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) sample buffer, boiled for 5 minutes, and
centrifuged at high speed for a few seconds to pellet debris, and
supernatants were loaded on a 10% SDS-PAGE.
Differentiation and analysis of K562 cells expressing FLAG-tagged
HOX A5.
pFAT- or pCMV-thy-1-transfected K562 cells were maintained in IMDM + 10% calf serum at less than 5 × 104 cells/mL.
Erythroid differentiation was induced by addition of 1 mmol/L sodium
butyrate. To measure heme content, aliquots of butyrate-treated cells
were removed from culture and stained with benzidine, as previously
described.16 Glycophorin A cell surface expression was
analyzed by FACS (Becton Dickinson) using specific monoclonal
antibodies according to the manufacturer's instructions (Dako,
Carpenteria, CA). Monocyte differentiation was induced by
the addition of 8 nmol/L 12-0-tetradecanoylphorbol 13-acetate (TPA;
Sigma). TPA-treated cells were cytospun onto slides and stained for
 -Napthyl acetate esterases according to the manufacturer's directions (Sigma).
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RESULTS |
Expression of HOX A5 in GM-CSF-stimulated bone marrow.
To characterize the pattern of HOX gene expression in normal human bone
marrow cells, progenitiors were stimulated with GM-CSF and harvested at
the earliest timepoint at which the biological effect of this cytokine
was discernable as a growth stimulus. To accomplish this, normal human
bone marrow cells were cultured for 4 days in semisolid medium
containing IMDM, 30% FCS, 0.9% methylcellulose, and 1 nmol/L GM-CSF.
Under these conditions, colonies of greater than 50 cells consisting of
mature neutrophils and/or monocytes form in 8 to 14 days. Clusters
containing 4 to 8 immature cells were picked on day 4 using a
micropipettor, pooled, and washed in PBS. Approximately 5 × 103 cells were harvested using this method. The timing of
this harvest is important, because it represents the earliest time at
which GM-CSF-stimulated cells can be identified by virtue of having divided and formed clusters. Although these cells are committed to the
myeloid lineage at the molecular level, they remain phenotypically undifferentiated.
To analyze the HOX gene expression in this limited amount of material,
we used an RT-PCR amplification protocol. mRNA and cDNA from these cell
clusters were generated using commercially available reagents (see
Materials and Methods). PCR amplification of cDNA was performed with
degenerate primers designed to recognize conserved sequences found in
members of the HOX family. The 3' primer (see Materials and
Methods) is directed against the WFQNRRMK amino acid coding sequence, a
highly conserved region overlapping the end of the -helix 3 of the
homeodomain. The 5' primer (see Materials and Methods) was
designed to recognize the cDNA code for the conserved pentapeptide
IYPWM sequence located upstream of the homeodomain of many of the HOX
genes.17 Because there is an intron situated between the
5' primer recognition sequence and the homeodomain containing the
3' primer recognition sequence, any genomic DNA contamination of
the sample would be indicated by the presence of products larger than
the expected 210-246 bp. A mock cDNA reaction control containing cell
cluster mRNA but no reverse transcriptase failed to generate a PCR
product of the same size, thereby confirming that the product seen was
not due to contamination with foreign DNA (data not shown).
The PCR reaction product was subcloned into the TA vector and
sequenced. This screen showed the expression of three members of the
HOX gene family, HOX A5, HOX B6, and HOX B7, in GM-CSF-stimulated human bone marrow. The 3' degenerate primer also appeared to have a high degree of affinity for the myeloperoxidase (MPO) cDNA; four
clones encoding its DNA were also found in the screen. The detection of
MPO in our samples provided a fortuitous control, because MPO mRNA is
only expressed early in granulocytic cell differentiation,18 thereby confirming that the harvested
cells were of early myeloid cell lineage. The expression and function of HOX B6 and HOX B7 during hematopoiesis was previously the subject of
study by our group and others.5,7,9,19 However, the role of
HOX A5 during hematopoiesis has not been extensively studied. We
therefore designed experiments to elucidate a function for the HOX A5
gene during blood cell development.
Expression of HOX A5 in GM-CSF-stimulated CD34+
cells.
Our data indicate that HOX A5 expression can be detected in a subset of
bone marrow cells that respond to GM-CSF stimulation, but it does not
address whether GM-CSF stimulation plays any role in the observed
expression of HOX A5. To investigate this, purified CD34+
cells from the bone marrow of a healthy human donor were cultured in
the presence or absence of 1 nmol/L GM-CSF in IMDM + 10% FCS for 72 hours. CD34 is a cell surface glycoprotein found on blood progenitor cells, including the pluripotent stem cell. Cells were then
harvested, with a portion plated into colony assays to verify the
viability and number of colony-forming cells and the remainder processed for analysis of HOX A5 expression. The colony assays (Fig 1A) showed that the presence or
absence of GM-CSF in the culture medium during the 72-hour incubation
did not make a significant difference in the viability or
differentiation potentials of the two populations, because both showed
the same pattern and number of BFU-E and CFU-GM formation. BFU-E and
CFU-GM are defined as progenitor cells commited to generate a colony of
mature erythroid or granulocyte/macrophage cells, respectively. To
perform semiquantitative RT-PCR, PCR primers were designed to recognize
sequences unique to the HOX A5 cDNA. The expected product from these
primers also spans an intron, so contaminating genomic DNA would be
recognized as a larger product. Amplification of cDNA from the
GM-CSF-treated and untreated populations with the HOX A5 primers was
followed by Southern blotting with a HOX A5-specific probe (Fig 1B),
which detected HOX A5 message in CD34+ cells before
treatment with GM-CSF. After treatment with GM-CSF, the level of HOX A5
expression in CD34+ cells decreased in two independent
experiments. Amplification of the same cDNAs with the
internal control GAPDH-specific primers, followed by densitometric
analysis of an ethidium bromide-stained gel containing their PCR
products, confirmed that the cDNAs from both populations were equal in
concentration and free of genomic DNA contamination. Titration
experiments show parallel linear increases in signal, in the range of
0.1 to 5 µg of input mRNA (Fig 1C).
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| Fig 1.
(A) No significant difference is seen in the
viability of CD34+ cells treated with or without GM-CSF
for 72 hours, as measured by colony-forming ability. (B) GM-CSF
treatment of CD34+ cells reduces expression of HOX A5
mRNA. cDNAs from GM-CSF-treated and untreated populations were
amplified with GAPDH- and HOX A5-specific primers. GAPDH products are
visualized by ethidium bromide staining, and HOX A5 products are
visualized by Southern blotting with a HOX A5-specific
32P-labeled probe. Controls containing mock cDNA reactions
are labeled as " RT." The diffuse signal below the HOX A5 band
is hybridization of the probe to the PCR primers. (C) Titration
experiment demonstrating the parallel linear increase in signal
relative to the concentration of input mRNA. Semiquantitative PCR was
conducted as described in Materials and Methods.
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These data are consistent with the findings of Sauvageau et
al,20 who reported the presence of HOX A5 in primitive
human CD34+, CD45RA ,
CD71, and CD38low bone marrow cells and
a decrease in its expression in a more mature subpopulation enriched in
granulopoietic clonogenic cells.
HOX A5 antisense ODN inhibit GM-CSF-stimulated colony formation.
Antisense ODN have proven to be a viable means by which to suppress the
expression of a gene of interest, and they can thereby serve as
valuable tools for the analysis of the biological function of a
specific gene.21 We and others used antisense ODN in colony assays to study homeobox gene function during
hematopoiesis.5,9,19,22 We used a similar approach to
analyze HOX A5 function during myelopoiesis. Antisense ODN to HOX A5
were designed to specifically recognize the HOX A5 mRNA (sequence in
the Materials and Methods). To confirm the activity of the ODN, K562
cells transduced to overexpress the full-length HOX A5 mRNA were
treated with 15 µmol/L antisense, sense, or nonsense ODN directed
against HOX A5 for 24 hours. HOX A5 mRNA expression of ODN-treated
cells was measured by HOX A5-specific semiquantitative RT-PCR. Relative
levels of total mRNA from each cell sample were measured by RT-PCR
amplification of GAPDH. Figure 2 depicts
the results of the densitometric scan of an ethidium bromide-stained
agarose gel containing HOX A5 and GAPDH RT-PCR products from each of
the ODN-treated cell samples. RT-PCR HOX A5 signal was normalized to
GAPDH signal. Antisense ODN-treated K562-LA5SN cells had 40% to 50%
of the level of expression found in untreated, sense, ODN- or nonsense
ODN-treated cells, and similar results were obtained in a second
independent experiment. Because these K562 cells have been engineered
to overexpress the HOX A5 gene, the levels of HOX A5 expression are
expected to be much higher than those found in normal bone marrow
cells.
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| Fig 2.
Antisense ODN to HOX A5 decrease levels of HOX A5 mRNA.
K562 cells transduced to overexpress HOX A5 are treated with 15 µmol/L of antisense, sense, nonsense (scrambled antisense), or no ODN
for 24 hours. Lanes 1 through 4 (+RT) contain cDNAs generated from
each treatment and were subjected to PCR amplification with HOX A5- and
GAPDH-specific primers. Lanes 6 through 10 (RT) contain HOX A5 and
GAPDH PCR products of mock cDNA reactions lacking reverse
transcriptase. Ethidium bromide-stained gels were digitally scanned,
and relative intensities of HOX A5 product bands compared with their
corresponding GAPDH bands are denoted below the lanes.
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In a separate experiment, 5.4 × 105 human bone marrow
CD34+ cells (78% positive) were cultured in X-vivo 10 defined medium in the presence of 15 µmol/L ODN for 2 days. mRNA from
each well was isolated and used to conduct semiquantitative RT-PCR
reactions for HOX A5 levels normalized to GAPDH. Similar to the results observed in K562 cells, the A5 signal in the antisense ODN-treated bone
marrow cells was 51% of the signal generated from cells treated with
nonsense ODN and 38% of the signal generated from diluent control-treated cells.
HOX A5 expression was detected in early myeloid progenitors harvested
as clusters. Antisense ODN were used to determine whether the
expression of HOX A5 plays a functional role in myeloid cell proliferation or maturation. Normal human bone marrow cells plated into
GM-CSF-containing colony assays demonstrated a specific dose-dependent decrease in the generation of colonies in the presence of HOX A5
antisense ODN compared with control ODN
(Fig 3A). In further experiments using a
single 20 µmol/L dose of ODN, granulocytic-monocytic colony formation
in the presence of HOX A5 antisense ODN was reduced nearly 80% (Fig
3B). These results show that HOX A5 expression is required for the
proliferation and maturation of CFU-GM, the bipotential progenitors of
mature granulocytes and monocytes.
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| Fig 3.
HOX A5 antisense ODN inhibit GM-CSF-stimulated colony
formation. (A) Human bone marrow cells plated into colony assays
containing 1 nmol/L human recombinant GM-CSF and increasing
concentrations of ODN. (B) A separate experiment similar to (A) at a
single dose of 20 µmol/L ODN. Data from both experiments are reported
relative to the number of colonies formed in assays containing no ODN
and are calculated from the mean of three assay wells.
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HOX A5 antisense ODN promotes the generation of BFU-E during ex vivo
expansion.
The GM-CSF colony assay results suggested an important biological role
for HOX A5 in determining lineage specificity during myelopoiesis. We
extended the analysis of the role of this gene by investigating its
actions in earlier hematopoietic progenitors. Our laboratory has
developed a system that supports the ex vivo expansion of CFU-GM and
BFU-E progenitors over a 10- to 14-day period.13 In this ex
vivo expansion system, CD34+ progenitor cells are cultured
in serum-free medium (GIBCO HSCM) containing IL-3, IL-6, G-CSF, and SCF
for a period of 10 days. CD34 is a cell surface glycoprotein found on
early hematopoietic progenitors and is routinely used for selection of
this cell population.23 During this culture, we observe a
considerable increase in nucleated cell number, along with limited
continuing generation of colony-forming progenitor cells. To gauge the
effect of HOX A5 suppression on the expansion of progenitor cells from
a pre-colony-forming cell, antisense, sense, or nonsense ODN to HOX A5
were included in the ex vivo expansion cultures. After 10 days in the
presence of ODN, the expansion cultures were plated into colony assays
containing IL-3, IL-6, G-CSF, SCF, and Epo without ODN to assay what
effect the presence of the ODN had on the expanding progenitors.
CD34+ cells cultured with HOX A5 antisense ODN during this
ex vivo expansion protocol (Fig 4)
displayed a dramatic and reproducible 100% increase in the number of
BFU-E colonies but no significant change in the numbers of CFU-GM. This
effect was specific for HOX A5 antisense ODN, because sense and
nonsense ODN showed no effect on expansion of progenitors. This
increase was apparent against a background of overall expansion of
BFU-E (Fig 5), suggesting that the increase
in the number of BFU-E seen at the end of the expansion is not merely
the result of the increased survival of erythroid progenitors during
expansion, but is due to an actual increase in their proliferation.
Thus, blocking the expression of HOX A5 in early hematopoietic
progenitors expands a population of cells with erythroid potential
without affecting the generation of granulocytic/monocytic
colony-forming cells. However, subsequent colony formation by these
CFU-GM is blocked by HOX A5 antisense ODN (Fig 3).
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| Fig 4.
Blocking HOX A5 expression increases the generation of
BFU-E. Ex vivo expansion cultures of CD34+ cells with HOX
A5 antisense, sense, or nonsense ODN were plated into colony assays
containing IL-3, IL-6, SCF, and G-CSF and 2 U Epo (see Materials and
Methods). Data are reported relative to the number of colonies
generated from nonsense ODN-treated cells and are representative of the
mean of three independent experiments, with each experiment containing
three assay wells.
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| Fig 5.
HOX A5 antisense ODN potentiates the expansion of BFU-E
during the ex vivo expansion of CD34+ cells. Fold
increase in the total numbers of colony types formed after ex vivo
expansion in the presence of HOX A5 antisense, sense, or nonsense ODN
compared with colony types present in colony assays plated at start of
expansion.
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Enforced expression of HOX A5 inhibits K562 erythroid
differentiation.
The multipotential cell line, K562, displays primitive erythroid
features and can be induced by sodium butyrate to differentiate into
cells expressing more mature erythroid markers such as
heme.24,25 Treatment of K562 cells with TPA has been
demonstrated to increase their expression of monocytic and
megakaryoblastic markers.26 To assess the effect of
overexpression of HOX A5 on hematopoietic differentiation, K562 cells
were transiently transfected with a vector expressing HOX A5 tagged
with the FLAG epitope (pFAT). This vector also expresses murine Thy 1.2 as a cell surface marker for selection of transfected cells via the
Miltenyi MiniMac system. Control K562 cells were transfected with the
vector expressing murine Thy 1.2 alone (pCMV-thy-1). Purification of
transfected cells via murine Thy 1.2 expression provided populations
containing at least 90% pFAT or pCMV-thy-1-transfected cells, as
determined by FACS analysis of Thy 1.2 staining (data not shown).
Western blot analysis using an anti-FLAG antibody confirmed the high
expression of the transfected FLAG-tagged HOX A5
(Fig 6) at the expected molecular weight of
36.4 kD.
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| Fig 6.
Expression of FLAG-tagged HOX A5 in K562 cells. K562
cells were transfected with FLAG-tagged HOX A5 (pFAT), empty vector
(pCMV-thy-1), or no vector (mock). Cells were purified via cell surface
expression of the transfection marker, murine Thy 1.2. Lanes labeled
"FT1" and "FT2" represent cell extracts from the
flow-through fraction of the two affinity columns used to select Thy
1.2-positive cells; lanes labeled "Thy 1.2+" are cell extracts
from the Thy 1.2-positive eluates from the columns; and the
lane labeled "FLAG-BAP" is FLAG-tagged bacterial
alkaline phosphatase (Eastman Kodak) used as a positve control for the
anti-FLAG Western antibody.
|
|
Overexpression of HOX A5 had no effect on TPA-induced monocytic
differentiation compared with control cells, as assayed by staining for
nonspecific esterases (-napthyl acetate;
Fig 7). This could be due to the fact that
TPA is a sufficiently strong inducer of monocyte differentiation such
that no further enhancement is seen with the addition of HOX A5.
Alternatively, other as yet unspecified factors may be limiting in the
cells that fail to differentiate. However, K562 cells
overexpressing HOX A5 exhibited only half the level of
butyrate-induced erythroid differentiation seen in
pCMV-thy-1-transfected cells (Fig 7). Additionally, a second marker of
erythroid phenotype, glycophorin A, was found to be significantly
diminished in K562 cells overexpressing HOX A5 (Fig 7). A time course
analysis shows that, even in transiently transfected K562 cells, HOX A5
expression limits erythyroid differentiation for 72 hours
(Fig 8).
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| Fig 7.
HOX A5 overexpression inhibits the butyrate-induced
differentiation of K562 cells. K562 cells were transfected with FLAG
epitope-tagged HOX A5 (pFAT) or the empty vector (pCMV-thy-1).
Erythroid differentiation was measured by heme content and cell surface
expression of glycophorin A 72 hours posttransfection. Heme content was
determined by direct benzidine staining of cells grown with or without
butyrate; the data represent a mean of seven experiments. Glycophorin A
was measured by FACS analysis and scored as the percentage of
transfected cells displaying specific fluorescence intensity above
background fluorescence levels; the data represent the mean of three
experiments. Monocytic differentiation was measured by nonspecific
esterase positivity of cells treated for 72 hours with or without 8 nmol/L TPA, and stained cytospun cells were scored by light microscopy;
the data represent the mean of two experiments. *P < .001;
**P < .05.
|
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| Fig 8.
Time course of butyrate-induced erythroid
differentiation. K562 cells were transiently cotransfected with
pCDNA3-F/A5.2 or the empty vector and pCMV-thy-1, as described in
Materials and Methods. Aliquots of cells were harvested at 24-hour
intervals, and heme content of cells without or with butyrate was
measured by benzidene staining.
|
|
| |
DISCUSSION |
In recent years, members of the homeobox family of genes have been
identified as important mediators of blood cell differentiation. The
expression patterns of these genes have been studied extensively in
cell lines of leukemic origin and, to a lesser degree, in normal hematopoietic cells (reviewed in Lawrence and Largman3).
Many of the studies of HOX gene function have been performed in cell lines of leukemic origin, in which expression of HOX genes may reflect
the cell line's transformed phenotype.3,10 Our work is
unique, in that it examines HOX gene expression at a fixed and tightly
defined moment in normal human myelopoiesis. Our results indicate that
at least three HOX genes, HOX A5, HOX B6, and HOX B7, are present in
normal human bone marrow progenitor cells early after a proliferative
response to GM-CSF. The function of HOX B7 during blood cell
development was the focus of one of our previous studies. We and others
demonstrated that HOX B7 expression is required for GM-CSF-stimulated
colony formation from both human and murine bone marrow5,9
and that downregulation of the gene is required for granulocytic
differentiation of HL-60 cells.5
Published reports indicate that HOX A5 expression appears to be
restricted to cells of myelomonocytic lineage and absent in cells of
erythroid lineage. Magli et al27 reported HOX A5 expression is present in HL-60 and U937 cells, which preferentially differentiate along the granulocytic-monocytic pathway, but is absent in the erythroleukemic cell lines, K562 and OCIM2. Vieille et al28 confirmed these findings in the same cells. Sauvageau et
al20 showed that the level of HOX A5 message in a
subpopulation of human CD34+ cells with erythroid potential
is lower than that found in more primitive cells or CD34+
cells displaying granulopoietic potential. The work presented here
complements these previous findings and defines a functional role for
HOX A5 in blood cell development. The increased generation of erythroid
progenitors seen when HOX A5 expression was reduced in the ex vivo
expansion assay suggests that HOX A5 may act as a repressor of the
generation or proliferation of erythroid progenitor cells. The decrease
in the erythroid phenotype of HOX A5-overexpressing K562 cells suggests
that the differentiation of an erythroid progenitor cell is also
repressed by HOX A5. Therefore, abrogation of HOX A5 expression would
allow for increased generation of erythroid progenitors and their
subsequent maturation. In another role, HOX A5 expression is present
and appears to be required for generation of mature progeny from CFU-GM
but not for production of the CFU-GM itself.
Our finding that HOX A5 expression appears to diminish in
CD34+ cells after GM-CSF stimulation supports a hypothesis
proposed by Lawrence et al29 that there is temporal
regulation of HOX gene expression during hematopoiesis, with high
expression of the widest range of HOX genes associated with uncommitted
progenitor cells and downregulation of the expression of certain HOX
genes being associated with differentiation. Their examination of human CD34+ progenitor cell subpopulations showed numerous HOX
genes expressed at a high level in the most immature cells and that
this level of expression was diminished in lineage-committed cells.
Other investigations have shown that HOX gene overexpression can lead to creation of hematopoietic cells with high proliferative activity or
leukemic phenotypes while displaying a decrease in
differentiation.6,30 During the ex vivo expansion assay,
expression of HOX A5 may help to maintain progenitor cells in an
uncommitted state. The removal of HOX A5 expression may have favored
the proliferation of a population of multipotential cells of a more
erythroid-committed subtype. Thus, understanding the means by which HOX
gene expression is suppressed may prove to be as important as
understanding how it is activated.
There is clear evidence that HOX genes regulate the expression of other
HOX genes in a self-referential pattern. Being transcription factors,
individual HOX genes likely bind directly to the promoter elements of
other HOX genes and regulate their transcription. Lobe31
demonstrated that exogenous expression of murine HOX A5 activated the
expression of numerous endogenous HOX genes, and HOX A5 has been shown
to bind to its own promoter.16 Second, translational
regulation of a HOX gene by another homeodomain protein could occur by
a mechanism recently described in which the homeodomain protein bicoid
binds the mRNA and acts as a translational repressor of the homeodomain
protein caudal in Drosophila embryos.32,33 This
interaction is postulated to be responsible for establishment of the
posterior-to-anterior concentration gradient of caudal seen during
Drosophila embryogenesis, and this gradient contributes to the
patterning of structures in the embryo. Finally, members of the murine
Pbx family of homeodomain proteins have been demonstrated to
heterodimerize with members of the murine HOX family, including HOX
A5,34 and this heterodimerization affects the DNA binding and transcriptional regulatory function of the HOX gene. Thus, regulated function of HOX genes could arise through competition for
heterodimerizing partners among the different HOX genes.
Whether HOX gene expression is directly influenced by cytokine
stimulation during hematopoiesis remains an open question. It has been
established that, during embryogenesis and in cell lines, polypeptide
growth factors and morphogens such as retinoic acid can regulate the
expression of homeobox genes.2 It is also possible that
cytokines may play no direct role in homeobox gene expression.
According to the stochastic model of hematopoiesis, lineage commitment
is a process that occurs randomly, and the role of a specific cytokine
is to prevent apoptosis and promote the proliferation of those cells
that express the receptor for that ligand, not to define what that
lineage may be (reviewed in Gordon and Amos35 and
Ogawa36). The observation that HOX genes are expressed at a
high level in very immature blood cells suggests that there may be a
high degree of competition between the HOX genes for DNA binding sites
and other molecular interactions. Disturbing this competitive balance
by a random change in the expression of specific HOX genes may provide
the underlying cause of lineage commitment.
We have defined an important role for the HOX A5 gene as a regulator of
myeloid cell proliferation and differentiation. Although we
demonstrated that manipulation of a single HOX gene can elicit profound
effects on these processes, it is likely that many different HOX genes
work together to fine-tune the output of blood cells produced.
Additionally, very little is known about the downstream effectors of
HOX genes. A more complete understanding of HOX gene function during
hematopoiesis awaits investigation of these issues.
| |
ACKNOWLEDGMENT |
The authors thank Dr Gay Crooks for providing the LA5SN vector; Dr
Michael Lill, Libby Walker, David Samuels, Maureen Lynch, and Negoita
Neagos for their help; and Wendy Aft for preparation of the manuscript.
| |
FOOTNOTES |
Submitted January 22, 1998; accepted January 20, 1999.
Supported by National Institutes of Health (NIH) SCOR Grant No. HL54850
(J.C.G.), NIH Grant No. R01 CA40163 (J.C.G.), and US Public Health
Service Award No. CA09056 (J.F.F.) from the National Cancer Institute.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Judith C. Gasson, PhD, Director, UCLA
Jonsson Comprehensive Cancer Center, 8-684 Factor Bldg, Box 951781, Los
Angeles, CA 90095-1781.
| |
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B. M. Owens and R. G. Hawley
HOX and Non-HOX Homeobox Genes in Leukemic Hematopoiesis
Stem Cells,
September 1, 2002;
20(5):
364 - 379.
[Abstract]
[Full Text]
[PDF]
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T. Taghon, F. Stolz, M. De Smedt, M. Cnockaert, B. Verhasselt, J. Plum, and G. Leclercq
HOX-A10 regulates hematopoietic lineage commitment: evidence for a monocyte-specific transcription factor
Blood,
February 15, 2002;
99(4):
1197 - 1204.
[Abstract]
[Full Text]
[PDF]
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Y. Yaron, J. K. McAdara, M. Lynch, E. Hughes, and J. C. Gasson
Identification of Novel Functional Regions Important for the Activity of HOXB7 in Mammalian Cells
J. Immunol.,
April 15, 2001;
166(8):
5058 - 5067.
[Abstract]
[Full Text]
[PDF]
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C. Buske, M. Feuring-Buske, J. Antonchuk, P. Rosten, D. E. Hogge, C. J. Eaves, and R. K. Humphries
Overexpression of HOXA10 perturbs human lymphomyelopoiesis in vitro and in vivo
Blood,
April 15, 2001;
97(8):
2286 - 2292.
[Abstract]
[Full Text]
[PDF]
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H. A. Golpon, M. W. Geraci, M. D. Moore, H. L. Miller, G. J. Miller, R. M. Tuder, and N. F. Voelkel
HOX Genes in Human Lung : Altered Expression in Primary Pulmonary Hypertension and Emphysema
Am. J. Pathol.,
March 1, 2001;
158(3):
955 - 966.
[Abstract]
[Full Text]
[PDF]
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J. A. Lekstrom-Himes
The Role of C/EBP{{varepsilon}} in the Terminal Stages of Granulocyte Differentiation
Stem Cells,
February 1, 2001;
19(2):
125 - 133.
[Abstract]
[Full Text]
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TOP
ABSTRACT
INTRODUCTION