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Leukemia (2002) 16, 1293–1301
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Expression pattern of HOXB6 homeobox gene in myelomonocytic differentiation and
acute myeloid leukemia
A Giampaolo1, N Felli2, D Diverio3, O Morsilli2, P Samoggia2, M Breccia3, F Lo Coco3, C Peschle2 and U Testa2
1
Department of Clinical Biochemistry, Istituto Superiore di Sanità, Rome, Italy; 2Department of Hematology and Oncology, Istituto Superiore
di Sanità, Rome, Italy; and 3Department of Cellular Biotechnology and Hematology, University La Sapienza, Rome, Italy
Homeobox genes encode transcription factors known to be
important morphogenic regulators during embryonic development. An increasing body of work implies a role for homeobox
genes in both hematopoiesis and leukemogenesis. In the
present study we have analyzed the role of the homeobox gene,
HOXB6, in the program of differentiation of the myeloid cell
lines, NB4 and HL60. HOXB6 expression is transiently induced
during normal granulocytopoiesis and monocytopoiesis, with
an initial induction during the early phases of differentiation,
followed by a blockade of expression at early maturation. The
enforced expression of HOXB6 in promyelocytic NB4 cells or
in myeloblastic HL60 cells elicited inhibition of the granulocytic
or monocytic maturation, respectively. Furthermore, HOXB6
was frequently expressed (18 out of 49 cases) in AMLs lacking
major translocations while it was expressed at very low frequency (two out of 47 cases) in AMLs characterized by
PML/RAR-␣, AML-1/ETO, CBF␤/MYH11 fusion and rearrangements of the MLL gene at 11q23. According to these observations, we suggest that a regulated pattern of HOXB6
expression is required for normal granulopoiesis and monocytopoiesis. Abnormalities of the HOXB6 expression may contribute to the development of the leukemic phenotype.
Leukemia (2002) 16, 1293–1301. doi:10.1038/sj.leu.2402532
Keywords: HoxB6; expression; myelomonopoiesis; differentiation;
leukemias
Introduction
Hematopoiesis depends on a complex series of cellular events
which requires both the maintenance of a pool of hematopoietic stem cells in an undifferentiated state and their maturation first to multipotent hemopoietic progenitors and then
to committed progenitors which first generate a progeny of
differentiated cells (hemopoietic precursors) and then the
mature cellular elements of blood (reviewed in Ref. 1). The
molecular mechanisms that lead to the generation of a progeny of progressively maturing cells starting from pluripotent
hemopoietic stem cells are poorly understood and begin only
in part to be elucidated.2
The process of hemopoietic differentiation seems to be
orchestrated at the molecular level by a series of transcription
factors which act according to a hierarchic pattern and following differentiation-specific pathways.2 Among these transcription factors a relevant role seems to be played by homeobox
(HOX) genes.
Products of HOX genes are localized in the nucleus and
represent a major class of transcription factors controlling
cellular proliferation/differentiation during early embryonic
development.3 Homeogenes, originally described in Drosophila melanogaster, are organized in gene clusters evolutionarly conserved.4,5 In humans the homeobox genes are
Correspondence: U Testa, Department of Hematology-Oncology, Istituto Superiere di Sanità, Viale Regina Elena, 299 Rome, 00161 Italy
Received 12 September 2001; accepted 20 February 2002
arranged in four gene clusters, named A, B, C and D, mapped
on chromosomes 7, 17, 12 and 2, respectively.6
Many of the HOX genes have been implicated in the regulation of both normal and leukemic hematopoiesis (reviewed
in Refs 7–10). It seems clear that HOX genes are expressed in
CD34+ cells and modulated according to stage and lineagespecific patterns. Particularly, HOXB genes on the 3′ cluster
side are highly expressed in primitive CD34+ populations9,11–14
and are colinearly expressed during differentiation of myeloid,
erythroid and lymphoid cells.12,13,15,16
HOX gene mutations associated with leukemias are not
known, but some leukemias are associated with irregular
expression of HOX genes due to translocation or abnormal
gene regulation. The HOX 11 gene is deregulated in T-ALL
with the t(10;14) translocation,17 while the HOX A9 is
involved in the t(7;11) translocation present in AML classified
as M2–M4 by French–American–British (FAB), that juxtaposes
the complete HOX A9 gene to a gene for nucleoporin
NUP98.18
Concerning HOXB6 expression, we have shown that this
homeobox is expressed in human hematopoietic progenitor
cells (HPCs) induced to differentiate into granulocytic lineage,
and that a blockade of its expression in HPCs specifically
inhibits granulomonocytic colony formation.13 However, the
HOXB6 gene could also be relevant for fetal erythropoiesis, as
suggested by studies in murine embryos.19 Finally, preliminary
observations based on the analysis of a few acute leukemia
patients showed the expression of HOXB6 in some acute
myeloid leukemias.20
In this study we have retrovirally transduced the HOXB6
gene in promyelocytic NB4 cells and in myeloblastic HL60
and investigated the effects of its enforced and deregulated
expression, showing an inhibition of the granulocytic and
monocytic maturation, respectively.
Since protein products resulting from chromosomal translocations occurring in the human acute leukemias can interfere
with regulated expression of genes that control normal hematopoiesis,21 including HOX genes, it seemed of interest to
evaluate the expression pattern of HOXB6 in acute myeloid
leukemias. HOXB6 mRNA was found to be expressed at variable levels in about 35% of acute myeloid leukemias classified as M1, M2, M4, M5 and M6 by FAB. By contrast, almost
all AMLs (45 out of 47) characterized by fusion genes underlying most common translocations (PML/RAR-␣, AML-1/ETO,
CBF␤/MYH11 and rearrangements of the MLL gene) showed
HOXB6 expression to be absent.
According to these observations we suggest that a regulated
pattern of HOXB6 expression is required for normal granulopoiesis and monocytopoiesis. Abnormalities of HOXB6
expression in acute leukemia may contribute to the development of the leukemic phenotype.
HOXB6 expression in myelomonocytic differentiation and AML
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1294
Materials and methods
Cloning of HOXB6 gene
Total RNA was extracted from human embryonal carcinoma
cell line NT2/D1 induced to differentiate by all-trans retinoic
acid (ATRA)10−5 m. The RNA extraction was performed by the
standard guanidinium thiocianate-CsCl technique. Two
micrograms of total RNA were reverse transcribed (RT) with
oligo (dT) as the primer. An aliquot of RT-RNA was amplified
using pfu DNA polymerase (Stratagene, La Jolla, CA, USA)
and the following primers: forward-primer CCCAATCTCGGAT
ATACTAC, reverse-primer CTCGGGTGGGGGAGCCAGGA.22
The PCR reaction mix was then directly ligated into the pCRScript Amp SK(+) (Stratagene) and used for bacterial transformation. A vector possessing the correct insert (674 bp) was identified and sequenced to confirm identity with published
sequence of HOXB6.22
Retroviral generation and infection of NB4 and HL60
cell lines
Briefly, the HOXB6 cDNA region encompassing the complete
coding sequence was subcloned at the BAMHI site of the
PINCO retrovirus.23 Viral supernatant was produced by transient transfection of Phoenix packaging cells with retroviral
construct as described in Ref. 23. NB4 and HL60 cell line
infection was obtained by three cycles of centrifugation of the
cells in viral supernatant at 1800 r.p.m. for 40 min in the
presence of 4 ␮g/ml of polybrene.
Hematopoietic growth factors and cell culture
Recombinant human growth factors were obtained from standard commercial sources.24,25 Iscove’s medium was freshly
prepared weekly.
Cell lines HL60 and NB4 were maintained in RPMI 1640
medium supplemented with 10% heat-inactivated fetal calf
serum (FCS).
Adult peripheral blood (PB) HPC purification and
clonogenic assay
HPCs were purified from PB buffy coat according to the
method previously reported24 and subsequently modified as
described.26
HPC granulopoietic (G) and monocytopoietic (M)
liquid suspension culture
Step IIIP HPCs were seeded (5 × 104 cells per ml) and grown
in liquid FCS− medium supplemented in G cultures with low
amounts of IL-3 (1 U/ml) and GM-CSF (0.1 ng/ml) and a saturating amount of G-CSF (10 ng/ml) as described;26 in M cultures with Flt3 ligand (100 ng/ml), IL-6 (10 ng/ml) and M-CSF
(10 ng/ml) as described.27 Cells were incubated in a fully
humidified atmosphere of 5% CO2/5% O2/90% N2 and were
periodically counted and analyzed for cell morphology, membrane phenotype and HOXB6 gene expression.
Leukemia
Reverse-transcription polymerase chain reaction (RTPCR) analysis
Total RNA was extracted by the standard guanidium thiocyanate-CsCl in the presence of 12 ␮g of Escherichia coli rRNA or
by the guanidium thyocyanate/acid phenol method28 technique.
RNA was reverse-transcribed (Moloney murine leukemia
virus; BRL, Gaithersburg, MD, USA) with oligo (dT) (BRL) as
a primer. The products were equalized by amplification of the
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or
human ribosomal protein S26 genes. cDNA from NT2/D1 teratocarcinoma cells induced to differentiate for 14 days with
ATRA 10−5 m29 was normalized together with normal and leukemic cell cDNAs: teratocarcinoma RT-RNA was used as
positive control in each PCR.
To evaluate HOXB6 expression, an aliquot of cDNA
(苲30 ng) was amplified within the linear range by 35 PCR
cycles (the cycle number was chosen on the basis of dose–
response curve for the NT2/D1 templates and analysis of aliquots of PCR reactions removed at different PCR cycles) using
the following primers: forward-primer CTTTGCCACTTC
CTCCTATTAC, reverse-primer CCAGACATACACACGTTAC
CAG. This amplification generated a 318 bp fragment of
human HOXB6 gene.22 PCR products were verified by Southern blotting utilizing an internal oligomer as a probe.
Evaluation of mRNA levels was performed by densitometric
analysis (Instant Imager, Packard, Canberra, Australia).
Cell surface marker and morphology analysis of HPC
cultures
To evaluate the differentiation of HPCs along the E and M
lineages the cells were stained with the following monoclonal
antibodies labeled either with FITC or PE: anti-CD34, antiCD15, anti-CD14 (Pharmingen, San Diego, CA, USA). Fluorescent cells were analyzed with a flow cytometer (FACS Scan,
Becton Dickinson, San Diego, CA, USA).
For cell morphology analysis cytospin preparations of cells
were performed at different days of culture and stained with
May–Grünwald-Giemsa. At least 200 cells were counted
and classified according to their differentiation/maturation
stage.
Cell surface and morphology analysis of NB4 and
HL60 cells
To evaluate the granulocytic differentiation capacities of both
NB4 cells transduced with the empty vector containing only
the reporter gene GFP (GFP NB4) or transduced with the
HOXB6 (HOXB6 NB4), these cells were grown in vitro for 5–
7 days in the presence of different doses of ATRA (from 10−9
to 5 × 10−7 m) and then analyzed for both cell surface markers
and cell morphology. For analysis of cell surface membrane
antigens the cells were labeled with the following PE-labeled
monoclonal antibodies: anti-CD11a, anti-CD11b, antiCD11c, anti-CD15, anti-CD18 and anti-CD54 (all from
Pharmingen). Fluorescent cells were analyzed as above and
the results are expressed in terms of both the percentage of
positive cells and mean fluorescent intensity, expressed in
arbitrary units.
To evaluate the monocytic differentiation capacities of
HL60 cells transduced either with the empty vector (GFP
HOXB6 expression in myelomonocytic differentiation and AML
A Giampaolo et al
1295
HL60) or with the HOXB6 gene (HOXB6 HL60), these cells
were grown for 4 days in the presence of 1␣-25OH-VitD3
(250 ␮g/ml) and then analyzed for cell surface markers and
cell morphology. For analysis of cell surface membrane antigens the cells were labeled with the following PE-labeled
monoclonal antibodies: anti-CD11a, anti-CD11b, antiCD11c, anti-CD18, anti-CD14 and anti-CD54 (all from
Pharmingen). Fluorescent cells were analyzed as above.
Leukemic patient samples
Cryopreserved cell samples from 96 patients with acute
myeloid leukemia, gathered from a single institution, were
studied and subclassified according to FAB nomenclature. The
original samples contained a range of 20 to 500 × 106 cells
each and ⬎80% blastic infiltration. Leukocytes were isolated
by Ficoll–Hypaque density centrifugation, lysed in guanidine
and freezed in liquid nitrogen. RNA was isolated by the guanidium thyocyanate/acid phenol method.28
All the samples were analyzed for the presence of
PML/RAR-␣, AML-1/ETO and CBF-␤/MYH11 fusion transcripts
according to the primers, protocols and criteria of the European BIOMED1 Concerted Action for standardization of minimal residual disease (MRD) studies in acute leukemia.30 All
assays were carried out using appropriate positive (cDNAs
from the NB4, KASUMI 1 and ME-1 cell lines) and negative
controls (all reagents + H2O). Controls as well as samples were
amplified in parallel with GAPDH primers, as internal control.
MLL rearrangements were evaluated by Southern blot analysis
using 10 ␮g of high molecular weight DNA electrophoresed,
blotted and hybridized with the appropriate 32P-labeled
probe.31
Figure 1
RT-PCR analysis of HOXB6 mRNA in purified HPCs differentiating along the granulocytic (a) or monocytic (b) lineages.
HOXB6 mRNA levels were investigated also in circulating neutrophils
(G) and monocytes (Mo), as well as in the positive control, ie ATRAinduced teratocarcinoma cells (C+).
Enforced expression of HOXB6 in NB4 promyelocytic
cells impaired granulocytic maturation
Statistical analysis
Given this pattern of HOXB6 expression during the normal
granulocytic and monocytic differentiation it seemed of inter-
Patient characteristics and complete remission rates were
compared using the chi-square test. Overall survival time was
calculated from the rate of diagnosis until death or at last follow-up examinations. Survival curves were estimated using
the product-limit method of Kaplan–Meier and were
compared using the log-rank square.
Results
HOXB6 expression during normal granulocytic and
monocytic differentiation
Using specific unilineage cell culture systems, allowing the
selective differentiation/maturation of purified HPCs along
either the granulocytic or monocytic lineages,25–27 we have
evaluated HOXB6 mRNA expression by RT-PCR during the
different stages of granulocytopoiesis and monocytopoiesis.
HOXB6 mRNA was only scarcely expressed in quiescent
HPCs and its expression markedly increased during the initial
stages of both granulocytic and monocytic differentiation,
with a peak of expression around day 5 of culture (Figure 1).
At later stages of differentiation/maturation, HOXB6 mRNA
progressively declined, being undetectable or detectable only
at very low levels in mature neutrophils and monocytes,
respectively.
Figure 2
Molecular map of PINCO/HOXB6 retroviral construct.
The two long terminal repeats (LTR), the Epstein–Barr virus nuclear
antigen-1 (EBNA-1) gene and the gene encoding for the enhanced
green fluorescence protein (GFP), controlled by a cytomegalovirus
promoter (CMV) are shown. The cloning sites (BamHI) are indicated.
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A Giampaolo et al
1296
est to evaluate a possible role of this gene in the control of
granulo–monocytic differentiation.
To evaluate this possible role we transduced with a retroviral vector expressing the HOXB6 gene (Figure 2) a leukemic
cell line capable of differentiating to granulocytes, such as
NB4 cells. The analogous vector containing GFP alone was
used as a control (GFP).
NB4 cell line did not display HOXB6 mRNA expression
detectable at the level of sensitivity of RT-PCR and RNAse
protection techniques (data not shown). In contrast, NB4 cells
transduced with HOXB6 gene under the control of a retroviral
promoter constitutively expressed HOXB6 mRNA, at a level
which remained constant during the differentiation ATRA
induced (Figure 3a).
We evaluated the capacity of GFP-NB4 as well as HOXB6NB4 cells to undergo granulocytic differentiation using ATRA
as a chemical inducer of neutrophilic maturation. GFP-NB4
cells exhibited morphological and phenotypical changes (a
marked enhancement of CD54, CD11b and CD18 expression)
typically associated with induction of granulocytic maturation, while these changes were only scarcely observed in
HOXB6 expressing NB4 cells (Figure 3b).
ATRA dose–response studies showed that at all doses of the
chemical inducer HOXB6-NB4 cells displayed a reduced
granulocytic maturation as compared to those observed in
GFP-NB4 cells, as evidenced by analysis of the levels of CD54
and CD18 achieved after treatment with different doses of
ATRA (Figure 4).
Figure 3
Expression of the granulocytic differentiation markers in NB4 cells transduced either with PINCO empty vector (GFP) or with
PINCO/HOXB6 (HOXB6) expressing vector and treated with ATRA (5 × 10−7 m). (a) HOXB6 transduced NB4 cells were analyzed for HOXB6
mRNA expression at various time point after ATRA induction. (b) Expression analysis of CD54, CD11b and CD18 differentiation markers in
GFP and HOXB6-NB4 cells. Mean ± s.e. of three independent experiments is shown.
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HOXB6 expression in myelomonocytic differentiation and AML
A Giampaolo et al
the large majority of HL60 cells (wt or transduced with the
empty vector) at consistently high levels; however, the CD14
antigen was induced only on about 50% of HOXB6 cells and
only at low levels (Figure 5c).
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HOXB6 expression in AML
Figure 4
Dose–response analysis of the effects of increasing concentrations of ATRA on CD54 and CD18 expression in GFP and
HOXB6-NB4 cells, analyzed at day 7. A representative experiment
is shown.
Constitutive HOXB6 expression in HL-60 cells
inhibited monocytic maturation
Since the pattern of HOXB6 expression during normal monocytopoiesis strictly resembles that observed during granulopoiesis, it seemed of interest to evaluate whether a constitutive
HOXB6 expression in HL-60 could impair the capacity of
these cells to undergo monocytic maturation in response to
treatment with 1␣25OH-VitD3. Wild-type HL-60 cells did not
express HOXB6 mRNA at detectable levels by RT-PCR and
RNAse protection (data not shown), while a high and constitutive HOXB6 expression was observed in HL-60 cells transduced with HOXB6 gene and such expression remained
unmodified after incubation of the cells with 1␣25OH-VitD3,
a chemical inducer of monocytic maturation (Figure 5a). After
4 days of treatment with this inducer wild type (wt) as well as
HL-60 cells transduced with the empty vector (GFP)
underwent monocytic maturation according to morphological
and phenotypical criteria. However, these changes were greatly inhibited in HL60 cells expressing the HOXB6 gene
(Figure 5b). The analysis of the kinetics of induction of CD14,
a membrane antigen specifically expressed in monocytes,
showed that this antigen was induced by 1␣25OH-VitD3 on
Given these effects of HOXB6 on granulocytic and monocytic
maturation it seemed of interest to evaluate HOXB6
expression on leukemic blasts derived from AML patients.
Ninety-six cases of AML, subdivided according to the FAB
classification, were screened for HOXB6 expression.
Interestingly, a consistent proportion (18/49) of AMLs lacking major translocations were found to be HOX B6+ (ranging
from 22 to 60% of AMLs classified as M1, M2, M4 M5 and
M6 by FAB) (Table 1); the cases expressing the highest levels
of HOXB6 mRNA were found among M2 and M4 leukemias (Figure 6).
By contrast, almost all AMLs (45/47) characterized by fusion
genes underlying most commons translocation showed HOX
B6 expression to be absent (Table 2). In particular, leukemic
blasts from 15 cases of M3 PML/RAR-␣+ were constantly
HOXB6 negative (Table 2); similarly, HOXB6 expression was
undetectable in 12 cases of AML with t(8;21), AML-1/ETO+,
in 10/11 cases of AML with inversion of chromosome 16,
CBF␤/MYH11+, and in eight out of nine AML with MLL
rearrangements. The frequency of HOXB6 expression was significantly (P ⬍ 0.001) different in the two AML groups with or
without major translocations.
M3 leukemic blasts tested negative for HOXB6 expression
even after in vitro treatment with ATRA, which downregulates
PML/RAR-␣ expression and induces granulocytic maturation
of these cells (data not shown).
Clinical outcome according to HOXB6 expression was
investigated in the group of 49 patients with normal karyotype.
Patients with positive HOXB6 expression and patients with
negative HOXB6 expression had essentially identical complete remission rates of 66.6% and 62.7% (P = 0.43). There
was a trend towards longer median survival in patients lacking
HOXB6 expression: median survival 26 months (range 1–86)
vs median survival of 15 months (range 1–84); however, this
difference did not achieve statistical significance (P = 0.24).
Discussion
In recent years, several members of the homeobox gene family
have been identified as important regulators of hemopoietic
cell differentiation. The expression patterns of these genes
have been extensively studied in cell lines and, to a less
extent, in normal hemopoietic cells (reviewed in Refs 9 and
10). Several of the HOX genes exhibited a temporal pattern
of expression, with a peak associated with the early stages of
HPC differentiation and downregulation of several HOX genes
being associated with later differentiation.12,13
HOXB6 is a prototype of these HOX genes, its expression
being restricted to the early phases of HPC differentiation (Ref
13 and the present paper), with downmodulation at
intermediate/late stages of maturation.
Given this peculiar pattern of expression it seemed of interest to evaluate the role of this HOX gene in granulocytopoiesis
and in monocytopoiesis. Enforced expression of HOXB6 in
NB4 and HL60 cell lines, which do not express HOXB6, elicLeukemia
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A Giampaolo et al
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Figure 5
Effect of HOXB6 enforced expression in HL60 cells on the monocytic maturation induced by 1␣25OH-VitD3 (250 ␮g/ml). (a)
HOXB6 transduced HL60 cells were analyzed for HOXB6 mRNA expression during monocytic differentiation. (b) Left: analysis of the expression
of CD11b, CD18, CD11a and CD14 in GFP and HOXB6 expressing HL60 cells treated for 4 days in the presence of 1␣25OH-VitD3. Right:
percentage of cells identified as mature monocytes in cells grown as above. Mean ± s.e. of three independent experiments is shown. (c) Kinetic
analysis of the expression of CD14 in wild-type, GFP and HOXB6-HL60 cells: the data are reported in terms of both the percentage of positive
cells (left) and of the mean fluorescence intensity (right). A representative experiment is shown.
Table 1
HOXB6 expression in acute myeloid leukemic samples
lacking major translocations
Sample
No.
FAB
HOXB6-positive sample No.
HOXB6-positive
frequency
1
2–10
11–23
24–33
34–44
45–49
M0
M1
M2
M4
M5
M6
—
2(+); 3(+)
11(++++); 12(++); 13(+); 14(+)
24(++); 25(+); 26(+); 27(±); 28(±)
34(+); 35(+); 36(+); 37(±)
45(+); 46(±); 47(±)
0/1
2/9
4/13
5/10
4/11
3/5
18/49
Expression was detected by RT-PCR analysis as shown in Figure 6.
Bands were quantitated by densitometric scanning of autoradiographs, using the value (+) when the band was similar to the positive control (ATRA-induced NT2/D1 teratocarcinoma cells). See
also Figure 6 and Materials and methods section.
ited a marked inhibition of granulocytic and monocytic maturation, respectively. The inhibition of ATRA-induced granulocytic maturation of NB4 cells is particularly interesting, as
retinoic acid is known to regulate homeobox gene expression,
including HOXB6, in other cell types.29 This inhibitory effect
Leukemia
of HOXB6 on granulocytic and monocytic maturation
suggests that a silencing of this HOX gene is required for the
normal development of granulopoiesis and monocytopoiesis.
It is of interest that the effects of HOXB6 are different from
those previously reported for HOXB7 in the HL60 cellular system. In fact, overexpression of HOXB7 in these cells elicited
an inhibition of ATRA-induced granulocytic maturation, but
did not affect the monocytic maturation induced by 1␣25OHVitD3.32 However, at variance with HOXB6, HOXB7
expression was induced during VitD3-driven maturation of
HL60 cells.
Previous studies on HOXB6 have suggested a possible role
of this gene in the control of erythropoiesis. Thus, transfection
of the K562 erythroid cell line with a HOXB6 antisense construct induced an increase of erythroid features, while an
opposite phenomenon was observed when the cells were
transfected with a HOXB6 sense.11 Furthermore, it was shown
that during embryonic/fetal murine development HOXB6
expression is closely associated with the regulation of the
erythropoietic system.19 In the present study, we show that a
regulated pattern of HOXB6 expression is also required for
normal granulocytic and monocytic differentiation.
There is growing evidence indicating that HOX proteins
play a role in leukemic transformation (reviewed in Ref. 33):
(1) in AMLs, several HOXA, HOXB and HOXC genes are
HOXB6 expression in myelomonocytic differentiation and AML
A Giampaolo et al
1299
Figure 6
RT-PCR analysis of HOXB6 mRNA in leukemic blasts derived from AMLs lacking major translocations. Only positive cases are
shown, see also Table 1. C+ indicates ATRA-induced teratocarcinoma cells used as positive control. The expression pattern of HOXB6 gene
was confirmed in at least two other experiments.
Table 2
HOXB6 expression in acute myeloid leukemic samples
characterized by PML/RAR-␣, AML-1/ETO, CBF␤/MYH11 fusion and
rearrangements of the MLL gene at 11q23
Sample No.
1–15
16–27
28–38
39–47
Molecular
defect
PML/RAR␣
AML-1/ETO
CBF␤/MYH11
MLL/SOUTH
HOX B6-positive HOX B6-positive
sample No.
frequency
—
—
28(±)
39(+)
0/15
0/12
1/11
1/9
2/47
Expression was detected by RT-PCR analysis. Bands were quantitated by densitometric analysis (see also legend to Table 1).
expressed, while in lymphoid malignancies, HOXC genes are
preferentially expressed; (2) in rare cases of leukemia HOX
genes are mutated, such as the HOXA9 gene fused together
with the nucleoporin gene as the consequence of the t(7;11)
translocation.18
This is the first study which reports the pattern of HOXB6
expression in a large number of AMLs. We observed HOXB6
expression in about 35% of AMLs lacking major translocations, with some cases of M2 and M4 leukemia expressing
particularly high levels of HOXB6 mRNA (see Table 1 and
Figure 6). The expression pattern of HOXB6 in AMLs does not
seem to be related to the differentiation potential of the leu-
kemic blasts in that it was similarly expressed in different subtypes of AML, including M1, M2, M4, M5 and M6.
Furthermore, with the exception of only two cases, HOXB6
expression was found to be absent in AMLs positive for the
expression of leukemogenetic fusion proteins (such as
PML/RAR-␣, MLI-1/ETO, CBF␤/MYH11 and MLL gene
rearrangements). These AMLs account for about 40% of total
AMLs and for the large majority (⬎90%) of AMLs with known
oncogenetic molecular defects.21
Since it was previously reported that HOXC6, paralogue to
HOXB6, was expressed in AML-M3,34 we analyzed the
expression of this gene in all M3 blasts. We detected HOXC6
expression in three out 15 cases (data not shown).
In particular, M3 leukemias were constantly found to be
negative for HOXB6 expression, which remained absent following ATRA treatment. Reasons underlying this finding are
at present unclear to us. Nevertheless, it is of interest that also
in two previous studies HOXA935 and HOXA1036 were found
to be expressed in all types of AMLs, with the constant exception of M3 leukemias. Based on those observations and on the
results of the present study, it is tempting to suggest that HOX
gene expression is suppressed in M3 leukemias probably
through an aberrant signaling mediated by the PML/RAR-␣.37
HOX gene expression in several cell types is stimulated following activation of retinoic acid receptors;29 thus, it may be
hypothesized that an aberrant signaling through the RAR-␣
fused with PML suppresses the stimulatory effect of normal
RARs on HOX gene expression.
Whatever mechanism is involved in HOXB6 expression in
AMLs, this phenomenon does not seem to be related to
clinical outcome.
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A Giampaolo et al
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This striking difference among different types of AMLs suggests that HOXB6 expression in AMLs could be specifically
associated with leukemias whose molecular defects remain to
be determined.
Acknowledgements
The authors are indebted to Dr Alessandra Carè for critical
reading of the manuscript and helpful suggestions. We thank
Giuseppe Loreto and Alfonso Zito for photographic assistance.
This work was supported in part by grant ‘Homeobox genes
in adult hematopoiesis’ from Istituto Superiore di Sanità
and by grants from AIRC, MURST 70% and Ministero della
Sanità.
18
19
20
21
22
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