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HEMATOPOIESIS
From the Molecular Medicine and Gene Therapy, Institute
of Laboratory Medicine, Lund University Hospital, Lund, Sweden; and
Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver,
Canada.
Recent studies show that several Hox transcription factors are
important for regulation of proliferation and differentiation in
hematopoiesis. Among these is H0XA10, which is selectively expressed at
high levels in the most primitive subpopulation of human
CD34+ bone marrow cells. When overexpressed, H0XA10
increases the proliferation of early progenitor cells and can lead to
the development of myeloid leukemia. To study the effects of H0XA10 on
primitive hematopoietic progenitors in more detail, transgenic mice
were generated with regulatable H0XA10 expression. The transgenic mouse
model, referred to as tetO-HOXA10, contains the
H0XA10 gene controlled by a tetracycline-responsive element and a minimal promoter. Thus, the expression of H0XA10 is
inducible and reversible depending on the absence or presence of
tetracycline or its analog, doxycycline. A retroviral vector containing
the tetracycline transactivator gene (tTA) was used to
induce expression of the H0XA10 gene in bone marrow cells
from the transgenic mice. Reverse transcription-polymerase chain
reaction analysis confirmed regulatable H0XA10 expression in several
transgenic lines. H0XA10 induction led to the formation of
hematopoietic colonies containing blastlike cells and megakaryocytes.
Moreover, the induction of H0XA10 resulted in significant proliferative advantage of primitive hematopoietic progenitors (spleen colony-forming units [CFU-S12]), which was reversible on
withdrawal of induction. Activation of H0XA10 expression in
tet0-H0XA10 mice will therefore govern proliferation of
primitive myeloid progenitors in a regulated fashion. This novel animal
model can be used to identify the target genes of HOXA10 and better
clarify the specific role of HOXA10 in normal and malignant hematopoiesis.
(Blood. 2001;98:3301-3308) Hematopoiesis is a lifelong, dynamic process in
which a small number of pluripotent hematopoietic stem cells (HSCs)
residing in the bone marrow (BM), give rise to all the mature blood
cells of both myeloid and lymphoid origin.1-3 This
powerful proliferation and differentiation in hematopoiesis requires
tight control to supply the host with all the different types of blood
cells required for normal function. Much information has emerged on the
role of regulatory factors and cytokines affecting hematopoiesis and several studies have shown that among candidate genes involved in the
regulation are genes encoding various transcription factors (reviewed
by Ogawa4 and Orkin5).
HoxA10 belongs to a large family of transcription factors that share a
highly conserved DNA binding domain, the homeodomain. In mammals, 39 homeobox (Hox) genes are grouped together in 4 clusters, A
to D,6-8 and over the last few years, accumulating evidence has shown that the Hox proteins are important in the mechanisms controlling differentiation and proliferation of
hematopoietic cells.9 Several Hox genes from
the A, B, and C clusters are expressed in a distinct pattern during
hematopoiesis and aberrant Hox gene expression has also been
documented in different types of leukemia.10-15 Direct
effects of Hox gene function on hematopoiesis have been
shown in various studies where the expression of Hox genes
has been modulated by antisense oligonucleotides,16,17 gene disruption by homologous recombination,18 or by
overexpression.15,19-25 In mice, for example, the
overexpression of HoxB4 leads to selective expansion of primitive
progenitor cells without development of leukemia or other malignancies.
In contrast, overexpression of HoxB3, HoxB8, HoxA9, and HoxA10
eventually leads to myeloid leukemia after a latency period of several
months. This latency period can be shortened dramatically if the Hox
cofactors, Pbx1 or Meis1, are co-overexpressed with their Hox
partner.22,26-28 Other myeloid-specific effects of HOXA10
have also been documented in studies using constitutive overexpression
of HOXA10 in mouse hematopoietic cells.25 In addition to
these findings, expression of the H0XA10 gene is found in
all types of human acute myeloid leukemia (except promyelocytic leukemia) but not in lymphoid leukemia, further implicating an important role for H0XA10 as a regulator of myeloid progenitors.
Because overexpression of H0XA10 in murine hematopoietic cells leads to
increased proliferation of primitive myeloid progenitors and the
generation of blast cell colonies in vitro but does not lead to the
immediate development of leukemia in vivo,25 we decided to
ask whether we could use the H0XA10 gene to generate a
transgenic murine model with inducible and reversible proliferation of
primitive hematopoietic progenitors. To achieve this, we used the
tetracycline-transactivating system (tet-system), placing the
H0XA10 gene under the control of the minimal promoter from human cytomegalovirus (hCMV) fused to the tet operator sequences (tetO).29 BM cells from the transgenic mice
were transduced with a retroviral vector containing the tetracycline
transactivator (tTA) to activate H0XA10 expression. The results show
that regulatable short-term overexpression of H0XA10 alters the
commitment and colony-forming ability of clonogenic progenitor cells
and increases the proliferation of primitive spleen colony-forming unit
(CFU-S) progenitors. It is noteworthy that the proliferative
effects on primitive CFU-S progenitors are reversible on withdrawal of
HOXA10 induction. Therefore, a novel animal model has been generated where regulatable expression of H0XA10 can be used to induce
proliferation of primitive hematopoietic progenitors in vivo in a
reversible fashion. This model can be used for biologic studies of
normal and abnormal hematopoiesis and to identify hitherto unknown
transcriptional targets of HOXA10.
Generation and screening of transgenic animals
Retroviral vector production
Retroviral transduction of primary BM cells
In vitro clonogenic progenitor assay
CFU-S12 assay The NIT-GFP transduced BM cells derived from transgenic or control animals were injected into lethally irradiated (950 cGy, 110 cGy/min, 137Cs gamma rays) recipient C57BlxCBA mice either directly after retroviral cocultivation (day 0; d0) or after 7 days of culture (d7) in IMDM containing 30% FCStetfree, 1% BSA, 10 4  -ME, 2% SCCM, 5 U/mL hEpo, and 1.4 mg/mL G418 in
the presence or absence of 2 µg/mL doxycycline. The number of cells
that each mouse received was adjusted to give 8 to 15 macroscopic
colonies per spleen. The mice receiving doxycycline were given 0.2 mg/mL doxycycline plus 3% sucrose in the drinking water. Twelve days after injection, animals were killed and the number of macroscopic colonies on the spleen was evaluated. An aliquot of the d7 cultured cells was taken and used for preparations of total RNA (RNeasy, Qiagen)
for Northern blot analysis to verify the presence or absence of the RNA
transcripts of the H0XA10 transgene. The 742-bp
XhoI/DraI fragment was used as a probe. RNA (20 µg) was used for blotting (PerfectHyb-Plus, Sigma). In some cases,
well-isolated colonies were cut open with scissors and the cells were
either transferred to microscopic slides and stained with Wright-Giemsa
for morphologic analysis or lysed for isolation of DNA or RNA for
subsequent PCR or RT-PCR analysis, respectively.
Flow cytometry Flow cytometric analysis (FACS) was used to determine the level of GFP expression for measurements of transduction efficiency as well as measurement of inducibility and down-regulation of GFP expression in the absence or presence of doxycycline. Peripheral blood and, in some cases, BM cells, were treated with ammonium chloride (Stem Cell Technologies) prior to the FACS procedure. Results were analyzed with CellQuest software (Becton Dickinson, San Jose, CA).
Generation and characterization of HOXA10 transgenic mice The HOXA10 cDNA used in this study is complementary to a messenger RNA (mRNA), which represents the most abundant HOXA10 transcript in human BM cells.34 This cDNA was subcloned into the pUHG10-3 plasmid where it is placed under the control of the regulatable promoter, PhCMV*-1/tetO. The rabbit-globin intron and poly A sequence is placed downstream of the
HOXA10 gene to generate a stable mRNA from the transgene
(Figure 1A). The expression of the HOXA10 cDNA is therefore dependent
on the activation of PhCMV*-1/tetO by the tTA,
which is composed of the tet repressor and the activation domain of viral protein VP16 of herpes simplex virus.29
Transgenic mice bearing the tetO-HOXA10 construct were
generated and 19 founders were shown to be positive when examined by
PCR and Southern blot analysis. Nine founder mice transmitted the
tetO-HOXA10 transgene through germline. In these mice the
number of transgene copies ranged from a few to more than 50 as
estimated by Southern blot analysis (Figure 1B and Table
1).
HOXA10 is inducible in tetO-HOXA10 hematopoietic cells To identify which HOXA10 transgenic lines were optimal with regard to inducibility of HOXA10 expression in the hematopoietic system, we first asked whether there was any leakiness in the expression of the HOXA10 cDNA in peripheral blood cells from uninduced transgenic animals. Total RNA was isolated from peripheral blood cells and RT-PCR was performed. Five of the lines did not express HOXA10 RNA (no background), 3 showed low levels of the transcript, and one line had a somewhat higher expression level in the uninduced state (Table 1). Next, we asked whether the expression of HOXA10 could be induced in the presence of tTA and, furthermore, if it could be down-regulated in the presence of doxycycline. Figure 2 shows the retroviral vector (NIT-GFP) and the experimental design for induction of the transgene. Four days after treatment with 5-FU, BM cells were harvested from the tetO-HOXA10 mouse lines, prestimulated for 48 hours, and then cocultivated with NIT-GFP viral vector producer cells for an additional 48 hours. The NIT-GFP vector contains the neoR along with the tTA gene and the tetO-GFP, which acts as an inducible marker gene responsive to tTA. This results in a simultaneous expression of HOXA10 and GFP genes in the absence of doxycycline and down-regulation of both these genes in the presence of doxycycline. After transduction the cells were grown in selective medium containing G418 (1.4 mg/mL) for 5 days in the presence or absence of doxycycline (2 µg/mL). Through RT-PCR amplification of the transgene, the effect of doxycycline on the expression of HOXA10 could be monitored. Five lines showed high induction of HOXA10 expression, whereas the others had moderate to low induction (Figure 3 and data not shown). No direct correlation could be seen between levels of induction or leakiness and copy number of the transgene (Table 1). Three transgenic lines (2-5, 6-2, 17-3) were identified where the HOXA10 transgene was highly inducible, whereas no expression was detected in the uninduced state (no leakiness with or without tTA) in transgenic peripheral blood or NIT-GFP-transduced BM cells. These findings demonstrate that cells from these transgenic lines can be induced to express HOXA10 to high levels from a baseline of practically zero in the uninduced state.
Induction of HOXA10 expression leads to proliferation of blastlike colonies in vitro Because HOXA10 expression is inducible and can be regulated in BM cells derived from the tet0-HOXA10 transgenic mice, we wanted to analyze whether the induction would affect proliferation and differentiation of hematopoietic progenitors in vitro. The experimental design is described in Figure 2B. BM cells, harvested from tetO-HOXA10 transgenic mouse lines 2-5 and 17-3 and control C57BL/6xCBA mice, were transduced with the NIT-GFP vector and plated in methylcellulose cultures for myeloid colony formation in the presence or absence of doxycycline (2 µg/mL). The proportion of G418-resistant colonies following transduction was 19.2% ± 4.1% and 19.8% ± 4.8% for transgenic and control cells, respectively (values are means ± SD from 4 independent experiments). The plating efficiency was similar for both types of cells and was not affected by addition of doxycycline (about 50 colonies/1000 cells). However, when G418-resistant methylcellulose colonies were analyzed on the plates after Wright-Giemsa staining, a clear difference was observed between the groups. Approximately 30% of the transduced progenitors derived from the transgenic mice formed large colonies containing megakaryocytes and blast cells in the absence of doxycycline (Figure 4A,B). This colony type was not seen in the presence of doxycycline nor in the transduced colonies derived from control mice. As previously described, the appearance of this special colony type was accompanied by a lower frequency of the CFU-GM and CFU-Mix colonies.25 Another striking feature was the absence of unilineage macrophage colonies among transduced transgenic progenitors in the absence of doxycycline as compared to approximately 20% frequency of CFU-M colonies in the presence of doxycycline and in the control settings (Figure 4A). The effects of homeobox transcription factors have been shown to be concentration dependent during development,35-37 and the same phenomenon could apply to the hematopoietic system as well. In light of this, it is noteworthy that the expression levels of HOXA10 that can be transiently induced are more than sufficient to generate a striking proliferative effect in the most primitive progenitors (megakaryocyte-blast progenitors) and to influence commitment in less primitive progenitors.
HOXA10 expression can be regulated in vivo To verify whether the HOXA10 induction can be applied in vivo, 2 lines (2-5 and 17-3) were also tested for regulatable expression of HOXA10 and GFP in mice receiving transplants of NIT-GFP-transduced tetO-HOXA10 BM cells. Directly after the transduction, 106 cells were injected into lethally irradiated recipients. By adding or removing doxycycline from the drinking water of the mice, both HOXA10 and GFP expression in peripheral blood cells could be regulated as measured by RT-PCR and FACS analysis. Representative results from a recipient of transduced BM from the 2-5 line are shown in Figure 5. Induction of both GFP and HOXA10 expression was seen in the absence of doxycycline (d0). Doxycycline was then administrated for 7 days and the expression of both HOXA10 and GFP was clearly down-regulated (d7). Withdrawal of doxycycline led to up-regulation of both the genes when measured 12 days later (d19). These results show that the expression of HOXA10 can clearly be regulated in vivo by administration and withdrawal of doxycycline to the transgenic mice.
Induction of HOXA10 expression leads to increased proliferation of CFU-S12 progenitors Because blastlike colonies could be generated in methylcellulose on HOXA10 induction, we wanted to determine whether primitive myeloid progenitors (CFU-S12) could be stimulated to proliferate in a regulatable manner. Constitutive overexpression of HOXA10 in murine BM cells has been shown to increase proliferation of primitive CFU-S12 progenitors when cultured for 7 days after the retroviral transduction.25 Therefore, we transduced BM cells from the tetO-HOXA10 mice as well as wild-type control mice with the NIT-GFP retroviral vector. The transduced cells were injected into lethally irradiated recipients either directly after transduction or after 7 days in selective liquid medium (1.4 mg/mL G418) with or without doxycycline as shown in Figure 2B. The frequency of day 12 CFU-S colonies was similar for all groups when measured directly after the retroviral transduction (Table 2). However, induced expression of HOXA10 during the 7-day culture increased the day 12 CFU-S content of the culture approximately 3-fold as compared to transduced control cells or transgenic cells grown in the presence of doxycycline (Table 2 and Figure 6). The HOXA10 expression in the 7-day culture was verified by Northern blot analysis (Figure 6C). These findings demonstrate that transient induction of HOXA10 increases the proliferation of primitive myeloid progenitors with CFU-S12 biopotency.
HOXA10-induced proliferation of primitive myeloid progenitors is reversible To verify that the enhanced proliferation of primitive progenitors was a direct result of HOXA10 expression, we asked whether these effects could be reversed by down-regulating the expression of HOXA10 following the initial induction. For this, an aliquot was taken from the NIT-GFP-transduced tetO-HOXA10 BM cells that had been cultured for 7 days in selective medium in the absence of doxycycline and were used in the previously described CFU-S12 assay. These cells were split into 2 cultures for an additional 5 days where the HOXA10 expression was either down-regulated (+ doxycycline) or maintained in an induced state ( doxycycline). The cells were then
injected into lethally irradiated recipient mice and 12 days later the
spleens of these mice were harvested and the colonies enumerated. Mice
receiving cells with up-regulated HOXA10 expression had approximately
40% higher number of colonies in their spleens as compared with mice
that were injected with cells grown in the presence of doxycycline for
the last 5 days (Figure 7). These results
indicate a direct and reversible effect of induced HOXA10 expression on
the proliferation of primitive myeloid progenitors.
The development of inducible gene expression systems that function
well in vivo to demonstrate biologic effects has been problematic. However, the advent of the tetracycline system has changed the possibilities considerably because background levels are relatively low
and the inducibility high, often reaching several orders of magnitude
above background in tissue culture cells.29 Several reports have also demonstrated that the tetracycline system functions in vivo, including studies that demonstrate biologic
effects,29,38-43 for example, induction of reversible
acute B-cell leukemia by BCR-ABL144 and an inducible block
in transforming growth factor- Whether the levels of HOXA10 in this inducible model are sufficient to give rise to acute myeloid leukemia, as reported by Thorsteinsdottir and colleagues,25 is currently under investigation. In preliminary experiments, mice receiving transplants of NIT-GFP-transduced tetO-HOXA10 BM cells have been monitored for up to a year without signs of leukemic development (data not shown). However, the long latency period observed before the onset of the leukemia25 strongly argues for the need of a secondary mutation(s), and indeed, here the genetic background of the mice might play a role as well. Simultaneous overexpression of cofactors of HOXA10 might facilitate the leukemic progression and this might even prove to be necessary for the leukemia to appear if the levels of HOXA10 expression do not reach the threshold needed. Should this be the case, then these mice could provide an important background to screen for genes that can collaborate and cause myeloproliferation or leukemia. Another way to enhance the potential leukemic development is to sort for transduced tetO-HOXA10 BM cells before transplantation, thereby increasing the overall impact of induced HOXA10 expression. We are currently addressing these questions. The effects of HOXA10 and closely related transcription factors on
proliferation and differentiation of primitive hematopoietic progenitors have been documented, but the molecular mechanisms causing
these effects are still poorly understood. Although most downstream
target genes of Hox transcription factors remain elusive, a few
examples have been emerging. The target genes even include other
homeobox transcription factors, generating complex cross-regulatory and
autoregulatory pathways.46-49 Recent findings also suggest the involvement of Hox transcription factors in various other pathways;
that is, HoxA5 affects expression of p5350 and the progesterone receptor,51 and HoxA9 can interact with Smad
proteins to repress TGF- The availability of new transgenic animals, in which tTA expression is restricted to certain tissues or even lineages, will enable studies where HOXA10 can be induced in a tissue-specific manner. Therefore, the tet0-HOXA10 mouse should be of value to many investigators who aim to study the effects of HOXA10 on the development, differentiation, and proliferation of cells in vivo.
We would like to thank Kristina Sundgren and Eva Gynnstam for expert animal care, Lilian Wittman for help with animal experiments, Rusty Gage for providing the NIT-GFP vector, and Sten Eirik Jacobsen and members of the Department of Stem Cell Biology, Lund University, for helpful advice and discussions.
Submitted April 30, 2001; accepted July 30, 2001.
Supported by grants from Cancerfonden, Sweden; Barncancerfonden, Sweden; Astra Draco (now AstraZeneca); and the Donation Funds Lund University Hospital (to S.K.); and by a clinical research award (ALF) from Lund University Hospital to S.K. and N.L. J.M.B. was supported by a graduate student award from the Medical Faculty, Lund University.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Stefan Karlsson, Molecular Medicine and Gene Therapy, Lund University Hospital, BMC A12, 221 84 Lund, Sweden; e-mail: stefan.karlsson{at}molmed.lu.se.
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Abstract
Introduction
