The oncoprotein E2A-Pbx1a collaborates with Hoxa9 to acutely transform primary bone marrow cells - PubMed (original) (raw)

The oncoprotein E2A-Pbx1a collaborates with Hoxa9 to acutely transform primary bone marrow cells

U Thorsteinsdottir et al. Mol Cell Biol. 1999 Sep.

Abstract

A recurrent translocation between chromosome 1 (Pbx1) and 19 (E2A) leading to the expression of the E2A-Pbx1 fusion oncoprotein occurs in approximately 5 to 10% of acute leukemias in humans. It has been proposed that some of the oncogenic potential of E2A-Pbx1 could be mediated through heterocomplex formation with Hox proteins, which are also involved in human and mouse leukemias. To directly test this possibility, mouse bone marrow cells were engineered by retroviral gene transfer to overexpress E2A-Pbx1a together with Hoxa9. The results obtained demonstrated a strong synergistic interaction between E2A-Pbx1a and Hoxa9 in inducing growth factor-independent proliferation of transduced bone marrow cells in vitro and leukemic growth in vivo in only 39 +/- 2 days. The leukemic blasts which coexpress E2A-Pbx1a and Hoxa9 showed little differentiation and produced cytokines such as interleukin-3, granulocyte colony-stimulating factor, and Steel. Together, these studies demonstrate that the Hoxa9 and E2A-Pbx1a gene products collaborate to produce a highly aggressive acute leukemic disease.

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Figures

FIG. 1

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 _Kpn_I (Kp) and _Eco_RI (E). LTR, long terminal repeat. G.F., growth factor.

FIG. 2

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).

FIG. 3

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.

FIG. 4

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 _Kpn_I 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.

FIG. 5

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 _Eco_RI, 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.

FIG. 6

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).

FIG. 7

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 _Eco_RI, 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.

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