INTRODUCTION

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Homeodomain (HD)-containing Hox gene products, regulators of pattern formation and tissue identity during embryogenesis (15), have also been identified as potential regulators of hemopoietic cell proliferation and differentiation (17). Several lines of evidence now also directly implicate Hox genes in human and murine leukemias. These include (i) the expression of Hoxa9 as a fusion protein Hoxa9-NUP98, in a subset of human myeloid leukemias (2, 21); (ii) the activation of Hoxa7 and Hoxa9 by retroviral insertional mutagenesis in myeloid leukemias in BXH-2 mice (22), and (iii) the development of leukemias in mice transplanted with bone marrow cells engineered to retrovirally overexpress Hoxb8, Hoxb3, Hoxa10, or Hoxa9 (13, 25, 32, 39).

A number of studies have demonstrated that Hox proteins collaborate in the in vitro DNA binding with a group of HD-containing proteins comprising Pbx and Meis families (34-36). This cooperative interaction between Pbx and Hox proteins is important since genetic and molecular studies in mice and in Drosophila have shown that Pbx1 (or its Drosophila homolog exd) is required for some of the biological functions of Hox proteins (1, 4, 19, 29, 30). The relevance of this Hox-Pbx interaction for malignant transformation has been demonstrated, as we recently showed that Hoxb3- or Hoxb4-induced transformation of Rat-1 fibroblasts is dependent on the presence of endogenous Pbx1 (14). An oncogenic collaboration between Hox and Meis proteins has also been established both by proviral insertion (22) and by retroviral overexpression studies (13). The Hox-Pbx interacting surfaces have been the focus of a number of studies and include, in addition to the HDs of both proteins, a tryptophan-containing motif located N terminal to the HD of Hox (found in several Hox proteins) and a region of 20 to 25 conserved amino acids called HCM (Hox cooperativity motif) located C terminal to the HD of Pbx1 (6, 24, 26, 27). The structure of the Hoxb1-Pbx1 complex bound to DNA was recently solved by crystallographic studies and confirmed the importance of these motives for Hox-Pbx1 interactions (28). Interestingly, these studies also demonstrated that the HCM motif of Pbx1 is part of its HD giving rise to a fourth  helix (28).

One member of the Pbx family, Pbx1, is also involved in human malignancy. In the t(1;19)(q23;p13.3) chromosomal translocation (11, 23), found in 10 to 20% of human pediatric pre-B acute lymphoblastic leukemias (3), most of the Pbx1 coding sequence, including the segment encoding the HD, is fused to the 5' half of the E2A gene, which encodes two transcription activation domains but lacks both the DNA binding and dimerization domains of E2A (11, 23). In addition to the involvement of the E2A-Pbx1 fusion gene in human acute lymphoblastic leukemia, various transformation assays have clearly demonstrated that the E2A-Pbx1 fusion protein is oncogenic. Transgenic mice expressing the E2A-Pbx1 cDNA in lymphoid cells developed T-cell lymphoblastic lymphomas (8), and mice reconstituted with bone marrow cells engineered by retrovirus-mediated gene transfer to overexpress E2A-Pbx1 developed growth factor-dependent acute myeloid leukemias (AML) (10).

The in vitro cooperative DNA binding properties of Pbx1 and E2A-Pbx1 with Hox proteins are not significantly different (18). This has lead to one current hypothesis, that at least in part, the transforming capacity of E2A-Pbx1 could be mediated by Hox gene products. In agreement with this possibility, deletion of all of the Pbx1 sequence from the E2A-Pbx1 fusion protein completely abrogates transformation of NIH 3T3 cells, indicating that the Pbx1 half of the fusion protein is essential for its transforming abilities (12, 20). Furthermore, the HCM of the Pbx1 half of the E2A-Pbx1 fusion protein is essential for cellular transformation induced by E2A-Pbx1 (5). This finding is very interesting in light of the recent crystallographic studies mentioned above which have redefined the HCM as part of an extended HD in Pbx1 (28). The concept that E2A-Pbx1-induced transformation is Hox dependent was challenged by studies which showed that the helices 1 to 3 of the HD in E2A-Pbx1 are dispensable for the capacity of this fusion protein to transform NIH 3T3 cells and T lymphocytes (12, 20). However, it was recently shown that the HD of E2A-Pbx1 is necessary to block cellular differentiation of myeloid progenitor cells, a process central to leukemic transformation (12). Together, these studies thus suggest that cellular transformation induced by E2A-Pbx1 may involve more than one pathway (mechanisms).

In contrast to E2A-Pbx1, the Pbx proteins lack inherent transforming potential (12-14, 20), and fusion with E2A is essential for the transforming ability of E2A-Pbx1. Structure-function and mutagenesis experiments have demonstrated that the two transcriptional activation domains in E2A are essential for mediating both the malignant transformation of NIH 3T3 cells (20) and blocking the differentiation of myeloid progenitor cells (12). Furthermore, using a Pbx-responsive sequence which allows cooperative DNA binding between Hox and Pbx1 (or E2A-Pbx1), it was shown in a reporter assay that E2A-Pbx1, but not Pbx1, could induce significant transcriptional activity of the reporter gene and that Hox proteins had the capacity to modulate the transactivating activity of E2A-Pbx1 (18). Together, these observations suggest that the oncogenic potential of E2A-Pbx1 is directly linked to the transcriptional activating function of the chimeric protein, and that this activity can be modulated by Hox gene products.

To examine whether Hox proteins and the E2A-Pbx1a fusion protein could collaborate to transform primary hematopoietic cells, we engineered mouse bone marrow cells, by retroviral gene transfer, to cooverexpress E2A-Pbx1a with Hoxa9, and the oncogenic collaboration between these two genes was directly tested in vitro and in vivo following transplantation of retrovirally transduced cells.

	MATERIALS AND METHODS

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Animals. All mice were originally bought from The Jackson Laboratory (Bar Harbor, Maine) and then bred and maintained in the specific-pathogen-free animal facility of the Clinical Research Institute of Montreal (IRCM). Donors of primary bone marrow cells were over 12-week-old (C57BL/6Ly-Pep3b × C3H/HeJ)F₁ [(PepC3)F₁] mice, and recipients were 7- to 12-week-old (C57BL/6J × C3H/HeJ)F₁ [(B6C3)F₁] mice. All animals were housed in ventilated microisolator cages and provided with sterilized food and acidified water.

Generation of recombinant retroviruses. The MSCV-Hoxa9-pgk-neo retroviral vector was described before (13). The human E2A-Pbx1a cDNA (a kind gift of Mark Kamps, San Diego, Calif.) was subcloned into the EcoRI site of MSCV-pgk-PAC retroviral vector (which confers puromycin resistance). Both MSCV vectors were kindly provided by R. Hawley (The Toronto Hospital Research Institute, Toronto, Ontario, Canada). High-titer helper-free retroviral producer cells were generated from GP+E-86 and BOSC-23 viral packaging cells and tested as reported previously (13).

Retroviral infection of primary murine bone marrow cells. Bone marrow cells obtained from (PebC3)F₁ mice injected 4 days earlier with 5-fluorouracil (50 mg/kg of body weight; Sigma) were prestimulated in the presence of interleukin-6 (IL-6), IL-3, and Steel factor and cocultivated on GP+E-86 viral producer cells engineered to produce the MSCV-Hoxa9-pgk-neo or the MSCV-E2A-Pbx1a-pgk-puro recombinant retrovirus. The procedure for cell harvesting, prestimulation, cocultivation, and bone marrow transplantation were performed as described elsewhere (13, 31). For double infections of bone marrow cells with Hoxa9 and E2A-Pbx1a, the respective viral producer cells were counted and seeded at equal numbers 24 h prior to the addition of bone marrow cells. Cells were cocultured for 48 h, with a medium change after 24 h. In an effort to enhance the efficiency of retroviral transduction of bone marrow cells, viral supernatants from Hoxa9- and E2A-Pbx1a-transfected BOSC-23 cells were added to the respective cocultures. The gene transfer efficiencies to primitive hemopoietic cells, as assessed by the in vitro colony formation of G418-resistant (containing the neo control or Hoxa9 retrovirus) or puromycin-resistant (containing the puro control or E2A-Pbx1a retrovirus) myeloid clonogenic progenitors, were 50, 40, 72, and 20% for the neo, puro, Hoxa9, and E2A-Pbx1a retroviruses, respectively. The efficiency of double infection of myeloid progenitor cells with the Hoxa9 and E2A-Pbx1a retroviruses was 4% as assessed by resistance of progenitor cells to both G418 and puromycin. All growth factors were used as diluted supernatants from appropriately transfected COS cells, as prepared at the IRCM. All reagents used in this study including media and serum were purchased at GIBCO Life Technologies.

Transplantations. The primary mice used in this study were generated by injecting intravenously into lethally irradiated (900 cGy, 179 cGy/min, ¹³⁷Cs gamma rays; J. L. Shepherd, San Fernando, Calif.) 7- to 12-week-old (B6C3)F₁ mice 2 × 10⁵ bone marrow cells derived from the (PepC3)F₁ mice immediately following their harvesting from the cocultivation with viral producer cells. To determine the transplantability of the diseases that developed in some of the primary mice, lethally irradiated or nonirradiated secondary (B6C3)F₁ recipients were injected with 10⁶ bone marrow and/or spleen cells obtained from the primary mice. For some of the leukemias that developed in the Hoxa9 + E2A-Pbx1a mice, the frequency of the leukemia repopulating cells (LRC) in bone marrow of these mice was determined by injecting various numbers of bone marrow cells (5 to 10⁶ cells per mouse) into lethally irradiated (B6C3)F₁ mice, along with a life-sparing dose of 10⁵ normal bone marrow cells derived from (B6C3)F₁ mice. LRC frequency in primary recipients was estimated by applying Poisson statistics to the proportion of negative recipients at different dilutions as described previously (37).

In vitro cultures. For myeloid clonogenic progenitor assays, cells were plated on 35-mm-diameter petri dishes (Corning, Fisher) in a 1.1 ml culture mixture containing 0.8% methylcellulose in alpha medium supplemented with 10% fetal calf serum, 5.7% bovine serum albumin, 10⁵ M -mercaptoethanol, 1 U of human urinary erythropoietin (Epo) per ml, 10% WEHI-conditioned medium (tested to contain 50 ng of IL-3 per ml), 2 mM glutamine, and 200 mg of transferrin per ml, in the presence or absence of 1.3 mg of G418 per ml and/or 1.5 µg of puromycin per ml. To test the ability of the retrovirally infected myeloid progenitor cells to form colonies in the absence of added growth factors, cells were plated in the above-described methylcellulose medium lacking both WEHI-conditioned medium and Epo. Bone marrow cells harvested from the cocultivation with virus-producing cells or recovered from reconstituted mice were plated at a concentration of 2 × 10³ to 8 × 10³ or 3 × 10⁴ cells/ml, respectively. Spleen cells from neo control mice were plated at a concentration of 3 × 10⁶ cells/ml, whereas those of the other reconstituted mice at 10⁵ cells/ml. Bone marrow or spleen cells from the reconstituted mice were plated at these same concentrations in the methylcellulose cultures lacking WEHI-conditioned medium and Epo. Colonies were scored on days 12 to 14 of incubation as derived from CFU-granulocyte-macrophage, burst-forming unit erythroid, or granulocyte-erythrocyte-macrophage-megakaryocyte CFU according to standard criteria (9). For some of the experiments, identification of colony types was confirmed by Wright staining of cytospin preparations of colonies. Bone marrow and/or spleen cells from the transplanted recipients were cultured in liquid cultures of Iscove's medium containing 10% fetal calf serum, 10⁵ M -mercaptoethanol, 2 mM glutamine, and 200 mg of transferrin per ml, in the presence or absence of 5 ng of IL-3 or 0.5 ng of granulocyte-macrophage colony-stimulating factor (GM-CSF) per ml.

Titration of growth factors. The presence of bioactive IL-3, G-CSF, and Steel present in conditioned medium obtained from 4-day-old cultures of cells obtained from the spleens of various mice and seeded at 10⁶ cells/ml was tested by [³H]thymidine incorporation assay to measure the proliferation of Ba/F3 (IL-3-responsive), NFS-60 (G-CSF- and IL-3-responsive) and TF-1 (Steel-responsive) cells. Titration of the bioactive substances was performed with COS cell-derived IL-3, G-CSF, and Steel as described above. The specificity of each growth factor to induce [³H]thymidine incorporation into the DNA of the appropriate cell line was verified by using antisera specific to IL-3 (50% neutralizing dose [ND₅₀] = 0.0015 to 0.025 µg/ml), G-CSF (ND₅₀ = 0.4 to 0.8 µg/ml), and GM-CSF (ND₅₀ = 0.05 to 0.15 µg/ml) (all from R&D). Complete and specific neutralization of [³H]thymidine incorporation was obtained for each supernatant tested except for that derived from 4.27 cells, where antibodies to both IL-3 and G-CSF were required for complete inhibition of proliferation of NFS-60 cells.

DNA and RNA analyses. To assess proviral integration, Southern hybridization analyses were performed as described elsewhere (13). Total cellular RNA was isolated with the TRIzol reagent, and Northern blot analysis was performed as described previously (7). The probes used were an XhoI/SalI fragment of pMC1neo (neo) (38), a HindIII/ClaI fragment of MSCV-pgk-PAC (puro), or the full-length 1.4-kb Hoxa9 and 2.9-kb E2A-Pbx1a cDNAs and were labeled with ³²P by random primer extension as described elsewhere (16). To assess the relative amounts of total RNA loaded, membranes were probed for 18S RNA by using end-labeled oligonucleotide 5'- ACG GTA TCT GAT CGT CCT CGA ACC-3'.

Nuclear extract preparation. A total of 2 × 10⁷ cells were washed twice in phosphate-buffered saline, incubated for 10 min at 0°C in 600 µl of lysis buffer (10 mM HEPES [pH 7.9], 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA), and then disrupted by passage through a 26-guage needle. Nuclei were collected by centrifugation at 500 rpm for 5 min and resuspended in 60 µl of extraction buffer (20 mM HEPES [pH 7.9], 400 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 5% glycerol) to allow elution of nuclear proteins by vigorous shaking for 1 h at 4°C. Nuclei were spun down by 2 min of centrifugation at 12,000 rpm, and the recovered supernatant was further cleared by 20 min of centrifugation at 40,000 rpm at 4°C. The protease inhibitors phenylmethylsulfonyl fluoride (2 mM), aprotinin (10 µg/ml), leupeptin (1 µg/ml), pepstatin A (10 µg/ml), and antipain (10 µg/ml) were added to both lysis and extraction buffers. Protein concentration was determined by the MicroBCA assay as recommended by manufacturer (Sigma, St. Louis, Mo.).

Western blot analysis. Thirty-microgram aliquots of proteins separated by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis were transferred to Immobilon P membranes (Millipore, Bedford, Mass.). Membranes were blocked with 5% nonfat milk in TBST (20 mM Tris-Cl [pH 7.6], 140 mM NaCl, 0.05% Tween 20) and then subjected to Western blot analysis with anti-E2A (E47; Santa Cruz Biotechnology Inc, Santa Cruz, Calif.) and anti-Hoxa9 (generously provided by H. Jeffrey Lawrence, San Francisco, Calif.). Bound antibodies were detected by using horseradish peroxidase-conjugated anti-rabbit antibody (Sigma) followed by enhanced chemiluminescence (Amersham, Buckinghamshire, England).

	RESULTS

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Altered colony formation and growth factor dependence of myeloid progenitor cells overexpressing Hoxa9 together with E2A-Pbx1a. The Hoxa9 and E2A-Pbx1a cDNAs were introduced downstream of the long terminal repeat of either the MSCVneoEB (Hoxa9) or MSCVpac (E2A-Pbx1a) retroviral vector (Fig. 1). Immediately following the infection of mouse bone marrow cells with the Hoxa9, E2A-Pbx1a, or Hoxa9 and E2A-Pbx1a retroviruses, the frequency of transduced myeloid colony-forming cells (CFC) was determined in methylcellulose cultures in the presence and absence of hematopoietic growth factors (Fig. 2A and B).

View larger version (12K): [in this window] [in a new window]   FIG. 1.   Structures of the retroviruses and overview of the experimental strategy used in this study. Diagrammatic representation of the integrated Hoxa9 and E2A-Pbx1a proviruses and the experiments described in this study. The expected sizes of the full-length viral transcripts are shown. Restriction sites indicated are KpnI (Kp) and EcoRI (E). LTR, long terminal repeat. G.F., growth factor.

View larger version (25K): [in this window] [in a new window]   FIG. 2.   Absence of differentiation and cytokine-independent growth in vitro characterizes myeloid CFC overexpressing both Hoxa9 and E2A-Pbx1a. (A) Total number of G418- or puromycin-resistant colonies generated per 104 bone marrow cells immediately following infection with neo control, puro control, E2A-Pbx1a, and Hoxa9 retroviruses, in the presence of added growth factors. No colonies grew in the absence of added growth factors. (B) Total number of G418- and puromycin-resistant colonies generated from 2 × 104 bone marrow cells immediately following infection with Hoxa9 and E2A-Pbx1a retroviruses, in the presence (black bars) and absence (gray bars) of added growth factors. Results shown in panels A and B represent the mean ± standard deviation of the number of G418 (neo and Hoxa9)- and/or puromycin (puro and E2A-Pbx1a)-resistant colonies detected on four different plates. (C) Well-isolated G418-, puromycin- or G418- and puromycin-resistant colonies were randomly picked (n = 16 to 20 for each infection type, from two different plates) on days 10 to 12 of incubation and examined by Wright staining. Colony types detected were classified as myeloid (mainly containing granulocytes and/or macrophages), myeloid-erythroid (containing granulocytes, macrophages, megakaryocytes, and erythrocytes), and immature (containing undifferentiated blast-like cells).

Cooverexpression of Hoxa9 with E2A-Pbx1a is acutely transforming. The capacity of Hoxa9- and E2A-Pbx1a-transduced bone marrow cells to induce acute leukemia in vivo was directly tested by transplanting Hoxa9- and E2A-Pbx1a-transduced bone marrow cells into lethally irradiated syngenic mice. In addition to the neo and puro controls, mice were also transplanted with bone marrow cells engineered to overexpress Hoxa9 or E2A-Pbx1a alone. To facilitate comparison of the dose of transduced cells transplanted in each group, the number of transduced myeloid progenitor cells (G418 and/or puromycin resistant) and the estimated number of long-term repopulating cells (LTRC) that were injected per mouse are shown in Table 1.

View larger version (142K): [in this window] [in a new window]   FIG. 3.   Cytopathological examination of hematopoietic and nonhematopoietic tissues from neo, Hoxa9, E2A-Pbx1a, and Hoxa9 + E2A-Pbx1a mice after Wright staining of peripheral blood smears (PB), bone marrow (BM) cytospins, and touch preparations of spleen (SPL), lung (LU), liver (LI), and brain (BR) tissues from representative neo control, E2A-Pbx1a, nonleukemic Hoxa9, and Hoxa9 + E2A-Pbx1a mice described in Table 2. Note the infiltration by immature blast cells in all six tissues of the Hoxa9 + E2A-Pbx1a mouse. In contrast, the spleen is the only tissue in the E2A-Pbx1a mouse that is infiltrated by such a cell type in addition to immature granulocytic cells. An increase in immature granulocytic cells is also detected in the spleen of the Hoxa9 mouse. Magnification, ×100 for all except the peripheral blood (×20). a, granulocyte; b, lymphocyte; c, blast; d, immature granulocyte; e, erythroblast; f, hepatocyte; g, erythrocyte.

View larger version (53K): [in this window] [in a new window]   FIG. 4.   Demonstration of the presence and expression of the integrated Hoxa9 and E2A-Pbx1a proviruses in primary mice. (A) Southern blot analyses of genomic DNA isolated from the bone marrow or spleen of the primary Hoxa9 + E2A-Pbx1a (top), E2A-Pbx1a (middle), and Hoxa9 (bottom) mice described in Table 2. DNA was digested with KpnI to release the integrated E2A-Pbx1a (5.8-kb) or Hoxa9 (4.1-kb) proviral fragment. The membranes were hybridized with a neo-specific probe to detect the Hoxa9 provirus and a puro-specific probe to detect the E2A-Pbx1a provirus. To provide a single-copy control of loading, the membranes were subsequently probed with full-length Hoxa9 cDNA probe. Note that the signal shown for the 8.0-kb single copy is that of endogenous Hoxa9. (B) Northern blot analysis of total RNA (10 µg) isolated from bone marrow or spleen cells of the primary Hoxa9 + E2A-Pbx1a (top), E2A-Pbx1a (middle), and Hoxa9 (bottom) mice described in Table 2. The membranes were hybridized with full-length E2A-Pbx1a or Hoxa9 cDNA probe. Each number assigned to a lane in panels A and B identifies a specific primary Hoxa9, E2A-Pbx1a, or Hoxa9 + E2A-Pbx1a mouse that is also identified with this same number in Fig. 5 or 7. GP-a9 or -EP, GP+E-86 Hoxa9 or E2A-Pbx1a viral producer cells.

Analysis of secondary recipients of leukemic bone marrow cells derived from Hoxa9 + E2A-Pbx1a mice. The acute leukemia induced by cooverexpression of Hoxa9 and E2A-Pbx1a was readily transplantable to secondary recipients, and clonal analysis of proviral integration sites demonstrated that the leukemias were derived from leukemic clone(s) present in the original donor leukemia (Fig. 5A). To determine the frequency of the biologically relevant cell in Hoxa9 + E2A-Pbx1a mice that can generate leukemia in secondary recipients (the leukemia repopulating cell [LRC]), bone marrow cells from Hoxa9 + E2A-Pbx1a mice 4 and 5 were injected at limiting dilution into lethally irradiated secondary recipients, along with a radioprotective dose of normal bone marrow cells. The frequency of the LRC was estimated at 1 in 124 and 1 in 60 bone marrow cells for Hoxa9 + E2A-Pbx1a mice 4 and 5, respectively (Fig. 5B). Interestingly, these studies also demonstrated a linear correlation between the number of LRC transplanted and the time required for the development of acute leukemia in the secondary recipients (Fig. 5C).

View larger version (63K): [in this window] [in a new window]   FIG. 5.   Clonal analysis of the acute leukemia that developed in primary and secondary Hoxa9 + E2A-Pbx1a recipients. (A) Southern blot analysis of DNA isolated from bone marrow of primary and secondary Hoxa9 + E2A-Pbx1a mice. The DNA was digested with EcoRI, which cuts the integrated provirus once, thus generating a unique fragment for each proviral integration site. The membranes were first hybridized with a neo-specific probe to detect Hoxa9 proviral fragments (top panel) and subsequently with a puro-specific probe to detect E2A-Pbx1a proviral fragments (bottom panel). Each primary recipient of Hoxa9- and E2A-Pbx1a-transduced cells is identified with a specific number shown in bold, and its secondary recipients are identified with derivatives thereof (1.1, 1.2, etc.). The number of leukemic cells transplanted per secondary recipients is indicated below each lane. For clarity, different leukemic Hoxa9- and E2A-Pbx1a-transduced clones detected in secondary recipients of mouse 4 are labeled from a to f; in secondary recipients of mouse 5, they are labeled a' and b'. (B) Evaluation by limiting dilution analysis of the frequency of the LRC in Hoxa9 + E2A-Pbx1a mice 4 and 5. Clonality of the majority of the secondary recipients used in this assay are shown in panel A. (C) Graphic display of the correlation between the number of LRC transplanted per secondary recipient of Hoxa9 + E2A-Pbx1a mouse 4 and the time needed for the development of leukemia in those recipients. The arrow on the x axis indicate the predicted time required for seven LRC (arrow on y axis) to give rise to overt leukemia in secondary mice (see Discussion). AL, acute leukemia; 2°, secondary; Cl, confidence interval.

Leukemic progenitor cells overexpressing Hoxa9 and E2A-Pbx1a do not require hematopoietic growth factors to proliferate in vitro. The leukemic cells in the bone marrow and spleen of Hoxa9 + E2A-Pbx1a mice formed colonies in methylcellulose cultures supplemented with growth factors with plating efficiencies of 2.4% ± 1.2% and 1.7% ± 0.2% for bone marrow and spleen cells, respectively, or with 8- and 2,800-fold increases compared to neo control mice (Fig. 6A and B). For the five mice tested, nearly 100% of their leukemic progenitor cells were resistant to both G418 and puromycin, demonstrating that these cells were all transduced with both Hoxa9 and E2A-Pbx1a. Interestingly, and in sharp contrast to bone marrow and spleen cells derived from nonleukemic Hoxa9 or E2A-Pbx1a mice sacrificed 56 and 51 ± 3 days after transplantation, the leukemic cells from Hoxa9 + E2A-Pbx1a mice had a similar capacity for colony formation in the presence or absence of added hematopoietic growth factors (Fig. 6A to C).

View larger version (31K): [in this window] [in a new window]   FIG. 6.   Growth factor-independent proliferation of leukemic blasts isolated from primary recipients of bone marrow cells overexpressing Hoxa9 and E2A-Pbx1a. The number of colonies generated from bone marrow (A) and spleen (B) cells of neo control, Hoxa9, E2A-Pbx1a, and Hoxa9 + E2A-Pbx1a mice, in the presence or absence of added growth factors, are shown. The results shown represent the mean ± standard deviation of the number of myeloid CFC in one femur and in the spleen of neo control (n = 2) and Hoxa9 (n = 3) mice at 56 days following transplantation and that of E2A-Pbx1a (n = 6) and Hoxa9 + E2A-Pbx1a (n = 6) mice when sacrificed as outlined in Table 2. (C) Microscopic view of colonies generated in methylcellulose cultures of spleen cells from E2A-Pbx1a and Hoxa9 + E2A-Pbx1a mice 13 days after initiation of the cultures. The arrows indicate the presence of small macrophage colonies that dominated the cultures from the E2A-Pbx1a mice. GF, growth factor. (D) Liquid culture of Hoxa9- and E2A-Pbx1a-overexpressing cells after 2 months of in vitro growth, demonstrating the growth of these cells in macroscopic clumps (magnification, ×10). The callous shows a Wright-stained cytospin preparation of the cells in these clumps, showing the immature morphology of these cells (magnification, ×100). (E) The presence of bioactive IL-3 and G-CSF in conditioned medium obtained from 4-day-old cultures of cells obtained from the spleens of a normal mouse, or from E2A-Pbx1a (n = 5) and Hoxa9 + E2A-Pbx1a (n = 6) mice and seeded at 106 cells/ml, was tested as outlined in Materials and Methods. (F) Western blot analysis of cell lysates from bone marrow (BM) and spleen (Spl) cells of normal mice and from one culture of Hoxa9 + E2A-Pbx1a leukemic cells. The membrane was probed with anti-E2A monoclonal antibody and then with polyclonal antisera directed against Hoxa9. The position of the 85-kDa E2A-Pbx1a protein and the 36-kDa Hoxa9 protein is indicated (arrows).

Overexpression of E2A-Pbx1a leads to the development of a highly polyclonal myeloproliferative syndrome (MPS). The recipients of Hoxa9-transduced bone marrow cells thrived normally for an extended period of time. At 56 days posttransplantation, three of these mice were sacrificed to allow comparison with the leukemic Hoxa9 + E2A-Pbx1a mice. At this time, hemopoietic regeneration by Hoxa9-transduced cells in these mice was polyclonal, similar to that detected in the neo control mice (Table 2 and Fig. 7A and B). The only hematological abnormality detected in these mice was in their spleens, which were slightly enlarged (Table 2), with some increase in cells with immature granulocytic morphology (Fig. 3) and myeloid CFC (Fig. 6A and B). As we previously reported (13), the remaining four Hoxa9 mice eventually developed mono- or biclonal AML with latency of 167 ± 32 days (Table 2 and Fig. 7B).

View larger version (61K): [in this window] [in a new window]   FIG. 7.   Southern blot analysis of DNA isolated from bone marrow of primary neo (A) and Hoxa9 (B) mice and primary and secondary E2A-Pbx1a mice (C). The DNA was digested with EcoRI, which cuts the integrated provirus once, thus generating unique fragments specific for the proviral integration site(s). The membranes were hybridized with a neo-specific probe to detect the neo control and Hoxa9 proviral fragments and a puro-specific probe to detect those of the E2A-Pbx1a provirus. Each primary mouse is identified with specific number in bold, and its secondary recipients are identified with derivatives thereof. All secondary recipients were transplanted with 1 × 106 to 2 × 106 bone marrow or spleen cells from the donor mouse. Where applicable, the time (in days posttransplantation [post-Tx]) for the development of acute leukemia in the secondary E2A-Pbx1a mice is shown. Asterisks denote secondary (2°) mice that developed MPS.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Several studies have suggested that cellular transformation induced by E2A-Pbx1 may result from collaborative interactions with products of the Hox gene complex. The studies described herein provided the first direct evidence demonstrating that E2A-Pbx1 collaborates with a Hox gene product (i.e., Hoxa9) and that this collaboration is sufficient to acutely transform primary bone marrow cells. Furthermore, bone marrow cells which coexpressed these two genes were able to proliferate in the absence of exogenous hematopoietic growth factors that, at least in part, can be explained by their production of IL-3 and/or G-CSF.

Cooverexpression of Hoxa9 and E2A-Pbx1a is sufficient to directly transform primitive hematopoietic cells. Three main observations in this study strongly suggest that cooverexpression of Hoxa9 and E2A-Pbx1a is sufficient to directly transform primitive bone marrow cells. These are (i) the ability of cooverexpression of Hoxa9 and E2A-Pbx1a to block differentiation and induce cytokine-independent growth of freshly infected primitive CFC (Fig. 2), (ii) the relatively short time required for the development of acute leukemia in the primary Hoxa9 + E2A-Pbx1a mice (39 ± 2 days), and (iii) the evidence that secondary recipients which receive a fixed number of Hoxa9- and E2A-Pbx1a-transduced LRC developed leukemia within a similar time frame (or even longer) as the primary recipients repopulated with comparable numbers of Hoxa9- and E2A-Pbx1a-transduced leukemic clones. For example, primary mouse 4, which had seven detectable leukemic clones (labeled a to f in Fig. 5A), exhibited full-blown leukemia in 40 days, whereas its secondary recipients of seven LRC would develop leukemia in 45 days (Fig. 5C). This finding suggests that the seven leukemic clones active in primary recipient 4 were already transformed when they were originally transplanted into this recipient.

E2A-Pbx1a enhances proliferation of committed progenitors in vivo. Recipients of E2A-Pbx1a-transduced bone marrow cells developed a polyclonal MPS characterized by large spleens and hind leg paralysis. With the exception of one mouse, this disease could not be transplanted to secondary recipients, suggesting that the target cell was not a stem cell but rather a more differentiated (and more frequent) short-lived progenitor cell. In support of this conclusion, the number of clones containing the E2A-Pbx1a provirus was much higher than the number of transduced stem cells injected per mouse (Table 1 and Fig. 7B). In addition, this MPS evolved to clonal AML in secondary recipients following a latency period which exceeded that required for disease to occur in primary mice, proving that E2A-Pbx1a alone was incapable of full leukemic transformation of stem cells.

Growth factor independence and production by Hoxa9- and E2A-Pbx1a-transduced bone marrow cells. Hoxa9- and E2A-Pbx1a-transduced bone marrow cells had the capacity to grow in the absence of growth factors when they were analyzed both immediately following retroviral gene transfer (Fig. 2B) and after being harvested from the bone marrow or spleens of leukemic mice (Fig. 6A to C). Except for bone marrow cells engineered to express either a signal-transducing kinase such as Tel-Jak2 (33) or an activated growth factor receptor (or a growth factor such as IL-3), we believe that these studies are the first to demonstrate the capacity of transcription factors to directly induce growth factor-independent proliferation of primary bone marrow cells (i.e., immediately following gene transfer [Fig. 2B]).