Meis1a suppresses differentiation by G-CSF and promotes proliferation by SCF: potential mechanisms of cooperativity with Hoxa9 in myeloid leukemia.

Hoxa9 and Meis1a are homeodomain transcription factors that heterodimerize on DNA and are down-regulated during normal myeloid differentiation. Hoxa9 and Meis1a cooperate to induce acute myeloid leukemia (AML) in mice, and are coexpressed in human AML. Despite their cooperativity in leukemogenesis, we demonstrated previously that retroviral expression of Hoxa9 alone--in the absence of coexpressed retroviral Meis1 or of expression of endogenous Meis genes--blocks neutrophil and macrophage differentiation of primary myeloid progenitors cultured in granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression of Meis1 alone did not immortalize any factor-dependent marrow progenitor. Because HoxA9-immortalized progenitors still execute granulocytic differentiation in response to granulocyte CSF (G-CSF) and monocyte differentiation in response to macrophage CSF (M-CSF), we tested the possibility that Meis1a cooperates with Hoxa9 by blocking viable differentiation pathways unaffected by Hoxa9 alone. Here we report that Meis1a suppresses G-CSF-induced granulocytic differentiation of Hoxa9-immortalized progenitors, permitting indefinite self-renewal in G-CSF. Meis1a also reprograms Hoxa9-immortalized progenitors to proliferate, rather than die, in response to stem cell factor (SCF) alone. We propose that Meis1a and Hoxa9 are part of a molecular switch that regulates progenitor abundance by suppressing differentiation and maintaining self-renewal in response to different subsets of cytokines during myelopoiesis. The independent differentiation pathways targeted by Hoxa9 and Meis1a prompt a "cooperative differentiation arrest" hypothesis for a subset of leukemia, in which cooperating transcription factor oncoproteins block complementary subsets of differentiation pathways, establishing a more complete differentiation block in vivo.

Estrogen-dependent E2a/Pbx1 myeloid cell lines exhibit conditional differentiation that can be arrested by other leukemic oncoproteins.

The molecular pathways of normal myeloid differentiation, as well as the mechanisms by which oncogenes disrupt this process, remain poorly understood. A major limitation in approaching this problem has been the lack of suitable cell lines that exhibit normal, terminal, and synchronous differentiation in the absence of endogenous oncoproteins and in response to physiologic cytokines, and whose differentiation can be arrested by ectopically expressed human oncoproteins. This report describes clonal, granulocyte-macrophage colony-stimulating factor-dependent myeloid cell lines that exhibit these properties. The cell lines were established by conditional immortalization of primary murine marrow progenitors with an estrogen-regulated E2a/Pbx1-estrogen receptor fusion protein. Clones were identified that proliferated as immortalized blasts in the presence of estrogen, and that exhibited granulocytic, monocytic, or bipotential (granulocytic and monocytic) differentiation on estrogen withdrawal. Differentiation was normal and terminal as evidenced by morphology, cell surface markers, gene expression, and functional assays. The differentiation of the cells could be arrested by heterologous oncoproteins including AML1/ETO, PML/RARalpha, PLZF/RARalpha, Nup98/HoxA9, and other Hox proteins. Furthermore, the study examined the effects of cooperating oncoproteins such as Ras or Bcr/Abl, which allowed for both factor-independent proliferation and differentiation, or Bcl-2, which permitted factor-independent survival but not proliferation. These myeloid cell lines provide tools for examining the biochemical and genetic pathways that accompany normal differentiation as well as a system in which to dissect how other leukemic oncoproteins interfere with these pathways.

HoxB8 requires its Pbx-interaction motif to block differentiation of primary myeloid progenitors and of most cell line models of myeloid differentiation.

HoxB8 was the first homeobox gene identified as a cause of leukemia. In murine WEHI3B acute myeloid leukemia (AML) cells, proviral integration leads to the expression of both HoxB8 and Interleukin (IL-3). Enforced expression of HoxB8 blocks differentiation of factor-dependent myeloid progenitors, while IL-3 co-expression induces autocrine proliferation and overt leukemogenicity. Previously, we demonstrated that HoxB8 binds DNA cooperatively with members of the Pbx family of transcription factors, and that HoxB8 makes contact with the Pbx homeodomain through a hexameric sequence designated the Pbx-interaction motif (PIM). E2a-Pbx1, an oncogenic derivative of Pbx1, both retains its ability to heterodimerize with Hox proteins and arrest myeloid differentiation. This observation prompts the question of whether E2a-Pbx1 and Hox oncoproteins use endogenous Hox and Pbx proteins, respectively, to target a common set of cellular genes. Here, we use four different models of neutrophil and macrophage differentiation to determine whether HoxB8 needs to bind DNA or Pbx cofactors in order to arrest myeloid differentiation. The ability of HoxB8 to bind DNA or to bind Pbx was essential (1) to block differentiation of factor-dependent myeloid progenitors from primary marrow; (2) to block IL-6-induced monocytic differentiation of M1-AML cells; and (3) to block granulocytic differentiation of GM-CSF-dependent ECoM-G cells. However, while DNA-binding was required, the HoxB8 Pbx-interaction motif was unnecessary for preventing macrophage differentiation of ECoM-M cells. We conclude that HoxB8 prevents differentiation by directly influencing cellular gene expression, and that the genetic context within a cell dictates whether the effect of HoxB8 is dependent on a physical interaction with Pbx proteins.

The HoxB1 hexapeptide is a prefolded domain: implications for the Pbx1/Hox interaction.

Hox proteins are transcriptional regulators that bind consensus DNA sequences. The DNA-binding specificity of many of these Hox proteins is modulated by the heterodimerization with partners, such as the Pbx proteins. This cooperative heterodimerization is accomplished through a conserved hexapeptide motif found N-terminal to the Hox DNA-binding homeodomain. Several human leukemias have been associated with a chromosomal translocation involving either the Hox gene (i.e., NUP98/HOXA9) or the gene encoding Pbx1 (E2A/PBX1). The transforming ability of these fusion oncoproteins relies at least partially on the ability to interact with one another through this hexapeptide motif. Herein we describe NMR structural calculations of the hexapeptide of HoxB1 (Nalpha-acetyl-Thr-Phe-Asp-Trp-Met-Lys-amide) that has been shown to mediate binding between HoxB1 and Pbx1 and a hexapeptide consensus sequence (Nalpha-acetyl-Leu-Phe-Pro-Trp-Met-Arg-amide). The consensus peptide exists in two conformations caused by cis-trans isomerization of the Phe-Pro peptide bond. The structures of the HoxB1 peptide and the trans form of the consensus peptide reveal a turn very similar to that found as part of the HoxB1/Pbx1/DNA complex in the X-ray crystal structure. This observation implies that this region is at least partially 'preformed' and thus ready to interact with Pbx1 and stabilize binding of Pbx1 and HoxB1 to DNA. The structural results presented here provide a starting point for synthesizing potential nonpeptide or cyclical peptide antagonists that mimic the interaction of these transcriptional cofactors resulting in a potential chemotherapeutic for certain types of leukemias.

Hoxa9 immortalizes a granulocyte-macrophage colony-stimulating factor-dependent promyelocyte capable of biphenotypic differentiation to neutrophils or macrophages, independent of enforced meis expression.

The genes encoding Hoxa9 and Meis1 are transcriptionally coactivated in a subset of acute myeloid leukemia (AML) in mice. In marrow reconstitution experiments, coexpression of both genes produces rapid AML, while neither gene alone generates overt leukemia. Although Hoxa9 and Meis1 can bind DNA as heterodimers, both can also heterodimerize with Pbx proteins. Thus, while their coactivation may result from the necessity to bind promoters as heterodimers, it may also result from the necessity of altering independent biochemical pathways that cooperate to generate AML, either as monomers or as heterodimers with Pbx proteins. Here we demonstrate that constitutive expression of Hoxa9 in primary murine marrow immortalizes a late myelomonocytic progenitor, preventing it from executing terminal differentiation to granulocytes or monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3. This immortalized phenotype is achieved in the absence of endogenous or exogenous Meis gene expression. The Hoxa9-immortalized progenitor exhibited a promyelocytic transcriptional profile, expressing PU.1, AML1, c-Myb, C/EBP alpha, and C/EBP epsilon as well as their target genes, the receptors for GM-CSF, G-CSF, and M-CSF and the primary granule proteins myeloperoxidase and neutrophil elastase. G-CSF obviated the differentiation block of Hoxa9, inducing neutrophilic differentiation with accompanying expression of neutrophil gelatinase B and upregulation of gp91phox. M-CSF also obviated the differentiation block, inducing monocytic differentiation with accompanying expression of the macrophage acetyl-low-density lipoprotein scavenger receptor and F4/80 antigen. Versions of Hoxa9 lacking the ANWL Pbx interaction motif (PIM) also immortalized a promyelocytic progenitor with intrinsic biphenotypic differentiation potential. Therefore, Hoxa9 evokes a cytokine-selective block in differentiation by a mechanism that does not require Meis gene expression or interaction with Pbx through the PIM.

Multiple effects of mercuric chloride on hexose transport in Xenopus oocytes.

HgCl(2) had both stimulatory and inhibitory effects on [(3)H]2-deoxyglucose (DG) uptake in Xenopus laevis oocytes. The Hg dose response was complex, with 0.1-10 microM Hg increasing total DG uptake, 30-50 microM Hg inhibiting, and concentrations >100 microM increasing uptake. Analyses of the effects of Hg on DG transport kinetics and cell membrane permeability indicated that low concentrations of Hg stimulated mediated uptake, intermediate concentrations inhibited mediated uptake, but high Hg concentrations increased non-mediated uptake. 10 microM Hg increased the apparent V(max) for DG uptake, but caused little or no change in apparent K(m). Phenylarsine oxide prevented the increase in DG uptake by 10 microM Hg, suggesting that the increase was due to transporter recruitment. Microinjecting low doses of HgCl(2) into the cell increased mediated DG uptake. Higher intracellular doses of Hg increased both mediated and non-mediated DG uptake. Both insulin and Hg cause cell swelling in isotonic media and, for insulin, this swelling has been linked to the mechanism of hormone action. Osmotically swelling Xenopus oocytes stimulated DG transport 2-5-fold and this increase was due to an increased apparent V(max). Exposing cells to 10 microM Hg or 140 nM insulin both increased cellular water content by 18% and increased hexose transport 2-4-fold. These data indicate that low concentrations of Hg and insulin affect hexose transport in a similar manner and that for both an increase cellular water content could be an early event in signaling the increase in hexose transport.

Granulocyte-macrophage colony-stimulating factor and B7-2 combination immunogene therapy in an allogeneic Hu-PBL-SCID/beige mouse-human glioblastoma multiforme model.

Glioblastoma multiforme is the most common primary central nervous system neoplasm. Its dismal prognosis has led to investigation of new treatment strategies such as immunogene therapy. We transduced the human glioblastoma cell line D54MG in vitro with genes encoding the proinflammatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), the T cell co-stimulatory molecule B7-2, or both (in a bicistronic vector) via retroviral vectors. Therapeutic gene expression by D54MG was high after transduction and selection (30 ng/10(6) cells/day for GM-CSF and > 2 orders of magnitude fluorescence shift on flow cytometry for B7-2). The effect of GM-CSF and/or B7-2 transduction on D54MG tumor growth in vivo was monitored in a novel allogeneic human peripheral blood lymphocyte-severe combined immunodeficiency mouse (Hu-PBL-SCID) model. GM-CSF- or B7-2-transduced tumors showed growth suppression in hu-PBL-reconstituted mice compared to untransduced and/or unreconstituted controls. Growth suppression was greatest for B7-2. Furthermore, vaccination with irradiated GM-CSF/B7-2-transduced tumor cells markedly inhibited growth of wild-type tumors at distant sites. Thus, this study illustrates a potential gene therapy strategy for glioblastoma multiforme patients using GM-CSF and/or B7-2 transduced tumor vaccines. Although extension of these allogeneic studies to an autologous system is critical, this is the first demonstration of in vivo efficacy of combination GM-CSF and B7-2 immunogene therapy for human glioblastoma multiforme.

Magnesium-dependent stimulation of protein synthesis by the insulin mimic, pervanadate.

The insulin mimic, peroxide of vanadate (pervanadate), stimulated 35S-methionine incorporation into Xenopus oocyte protein in a Mg(2+)-dependent manner. Reducing the extracellular Mg2+ concentration from 1.0 to 0.1 mM decreased the pervanadate-stimulated component of incorporation by 35%; with 0.01 mM Mg2+ or lower, the pervanadate-stimulated component was abolished. In addition, reducing extracellular Mg2+ to 0.01 mM inhibited about 50% of the insulin-stimulated component of methionine incorporation. Mg2+ depletion had no effects on incorporation in controls or when protein synthesis was stimulated by Zn2+ or bovine growth hormone. Thus, not all substances that stimulated protein synthesis showed a dependence on extracellular Mg2+. Reducing extracellular Ca2+ had no effects on methionine incorporation in control cells or in cells stimulated by pervanadate or insulin. When oocytes maintained in a paraffin oil medium were brought into contact with a 0.5 microliter droplet of buffer containing the Mg2+ indicator dye, mag-fura-2, and pervanadate, apparent droplet Mg2+ decreased rapidly, indicating net uptake by the cells. Insulin also caused a net uptake of Mg2+. In contrast, apparent extracellular Mg2+ was constant when cells were in contact with droplets containing no effectors. Together, these data indicate that extracellular Mg2+, but not Ca2+, is involved in the stimulation of protein synthesis by pervanadate, and to a lesser extent by insulin. Pervanadate appears to induce a net uptake of Mg2+, and this change in membrane transport may be an early event in signalling the increase in translation.

Multiple sites of vanadate and peroxovanadate action in Xenopus oocytes.

In Xenopus laevis oocytes, the insulin mimics, vanadate and peroxovanadates (PV), stimulated the uptake of 3H-2-deoxyglucose and incorporation of 35S-methionine into protein. For both hexose transport and protein synthesis, peroxovanadates (produced by reacting vanadate and H2O2) were at least as potent as vanadate. Microinjection of peroxovanadates into the oocytes stimulated 2-deoxyglucose uptake. However, methionine incorporation was not stimulated by microinjection of peroxovanadate or vanadate solutions. Consistent with these results and with the possibility that vanadate and peroxovanadates enter the cell on a phosphate transporter, raising the medium phosphate concentration from 1 mM to 10 mM blocked vanadate-stimulated hexose transport and partially reduced peroxovanadates stimulation of hexose transport. Increased medium phosphate did not reduce stimulation of protein synthesis by either effector. Taken together, these data indicate that vanadate/peroxovanadates act at both intracellular and extracellular sites. Action at the former stimulates hexose uptake and action at the latter, protein synthesis.

ATP-dependent transport of tetraethylammonium by endosomes isolated from rat renal cortex.

During renal organic cation secretion by some species, intracellular concentrations greatly exceed the 10- to 15-fold ratio predicted by the potential-driven mechanism thought to mediate their basolateral uptake. Free cytoplasmic organic cation concentrations within the tubular cells might be decreased through sequestration within intracellular organelles. The data reported here show that endosomal vesicles isolated from rat renal cortex take up tetraethylammonium (TEA) by an ATP-dependent mechanism. Addition of 0.2-5 mM ATP to the medium stimulated uptake 5- to 10-fold at 5 min and 20-fold at 60 min. More than 80% of the ATP-dependent uptake was associated with an osmotically active space. The nonhydrolyzable ATP analogue, adenosine 5'-O-(3-thiotriphosphate), did not stimulate TEA uptake. Mg2+ and Cl- were required for stimulation. Uptake was inhibited by several organic cations, including TEA itself. Uptake was also inhibited by inhibitors of intravesicular acidification, e.g., monensin and N-ethylmaleimide. Furthermore, the ATP requirement could be bypassed by establishing a pH gradient (inside acidic). These data show that endosomal TEA accumulation is mediated by proton/TEA exchange and is driven by the pH gradient maintained by H(+)-adenosinetriphosphatase. This potent sequestration mechanism may play an important role in organic cation secretion.

Stimulation of protein synthesis by internalized insulin.

Previous studies showed that microinjected insulin stimulates transcription and translation in Stage IV Xenopus oocytes by acting at nuclear and cytoplasmic sites (Miller, D.S., 1988, 1989). The present report is concerned with the question of whether hormone, internalized from an external medium, can act on those sites to alter cell function. Both intracellular accumulation of undegraded 125I-insulin and insulin-stimulated 35S-methionine incorporation into oocyte protein were measured. Anti-insulin antiserum and purified anti-insulin antibody were microinjected into the cytoplasm of insulin-exposed cells to determine if insulin derived from the medium acted through internal sites. In cells exposed for 2 h to 7 or 70 nM external insulin, methionine incorporation was stimulated, but intracellular hormone accumulation was minimal and microinjected antibody was without effect. In cells exposed for 24 h, methionine incorporation again increased, but now accumulation of undegraded, intracellular hormone was substantial (2.6 and 25.3 fmol with 7 and 70 nM, respectively), and microinjected anti-insulin antibody significantly reduced the insulin-stimulated component of incorporation; basal incorporation was not affected. For cells exposed to 70 nM insulin for 24 h, inhibition of the insulin-stimulated component was maximal at 39%. Thus under those conditions, about 40% of insulin's effects were mediated by the internal sites. Together, the data show that inhibition of insulin-stimulated protein synthesis by microinjected antibody was associated with the intracellular accumulation of insulin. They indicate that when oocytes are exposed to external insulin, hormone eventually gains access to intracellular sites of action and through these stimulates translation. Control of translation appears to be shared between the internal sites and the surface receptor.