The FLT3 tyrosine kinase receptor inhibits neural stem/progenitor cell proliferation and collaborates with NGF to promote neuronal survival.

The FLT3 receptor tyrosine kinase (FLT3) was originally identified on hematopoietic stem cells (HSCs) and its ligand (FL) induces HSC proliferation. As stem cells originating from various tissues are more similar than once thought, the goal of this study was to determine whether neural stem cells express FLT3 and proliferate in response to FL. In fact, a subset of neural stem/progenitor cells does express FLT3, but contrary to our expectations, FL inhibited EGF and FGF-2 stimulated proliferation. Since FLT3 is expressed weakly by proliferative neuroepithelia but strongly by subsets of neurons in the CNS and PNS, we tested its ability to support neuronal survival. FL synergized with NGF to promote the survival of cultured DRG neurons, although it lacked any neurotrophic activity alone. We conclude that FL serves as an adjunct trophic factor in the nervous system, which differs from its role in the hematopoietic system.

cDNA cloning, chromosomal mapping, and expression analysis of human VE-Cadherin-2.

Murine vascular endothelial cadherin-2 (VE-cad-2) is a cellular adhesion molecule that is distinct from vascular endothelial cadherin 1 (VE-cad-1) in that it does not interact with catenins and does not appear to affect cell migration or growth. In this study, we have cloned a full-length cDNA of the human homolog of VE-cad-2 and used it to map the chromosomal locus of the VE-cad-2 gene. Human VE-cad-2 maps to Chromosome (Chr) 5q31. The cDNA of human VE-cad-2 is highly homologous to mouse VE-cad-2, except for a C-terminal tail. The genomic structure of VE-cad-2 is strikingly similar to that reported for a large family of neuronal protocadherin genes mapped to Chr 5q, yet the amino acid sequences between VE-cad-2 and the protocadherins are substantially divergent. The promoter of human VE-cad-2 contains two TATA boxes and transcription initiates from a single site 3' to these elements. Similar to mouse VE-cad-2, the human gene is expressed primarily in highly vascularized tissues.

Identification of the residues in the extracellular region of KDR important for interaction with vascular endothelial growth factor and neutralizing anti-KDR antibodies.

The kinase domain receptor (KDR) of vascular endothelial growth factor (VEGF) is the main human receptor responsible for the angiogenic activity of VEGF. The extracellular region of KDR is comprised of seven immunoglobulin-like domains, of which the first three have been shown to be required for ligand binding. We have previously described antibodies directed against the extracellular region of KDR, including MAB383 and MAB664, which were shown to block the binding of VEGF to the receptor and to inhibit both VEGF-induced mitogenesis of human endothelial cells in vitro and tumor growth in vivo. Here we generated a series of KDR deletion mutants consisting of truncated extracellular regions and mapped out the domain(s) responsible for binding to VEGF and the neutralizing anti-KDR antibodies. All neutralizing antibodies were found to require domain 3 for efficient binding. Alanine-scanning mutagenesis of domain 3 identified two different sets of five residues, Ile(256), Asp(257), Glu(261), Leu(313), and Thr(315) and Tyr(262), Pro(263), Ser(264), Ser(265), and Lys(266), that were critical for binding to MAB383 and MAB664, respectively. Combination of alanine mutations affecting both MAB383 and MAB664 binding resulted in a variant that also lost binding to VEGF. These results suggest that the residues within this region of domain 3 are critical for VEGF binding. Our studies provide a basis for the mechanism of action of our anti-KDR antibodies and establish a functional foundation for the development of other classes of antagonists to the receptor.

Antivascular endothelial growth factor receptor (fetal liver kinase 1) monoclonal antibody inhibits tumor angiogenesis and growth of several mouse and human tumors.

Tumor angiogenesis is mediated by tumor-secreted angiogenic growth factors that interact with their surface receptors expressed on endothelial cells. Vascular endothelial growth factor (VEGF) and its receptor [fetal liver kinase 1 (Flk-1)/kinase insert domain-containing receptor] play an important role in vascular permeability and tumor angiogenesis. Previously, we reported on the development of anti-Flk-1 and antikinase insert domain-containing receptor monoclonal antibodies (mAbs) that potently inhibit VEGF binding and receptor signaling. Here, we report the effect of anti-Flk-1 mAb (DC101) on angiogenesis and tumor growth. Angiogenesis in vivo was examined using a growth factor supplemented (basic fibroblast growth factor + VEGF) Matrigel plug and an alginate-encapsulated tumor cell (Lewis lung) assay in C57BL/6 mice. Systemic administration of DC101 every 3 days markedly reduced neovascularization of Matrigel plugs and tumor-containing alginate beads in a dose-dependent fashion. Histological analysis of Matrigel plugs showed reduced numbers of endothelial cells and vessel structures. Several mouse tumors and human tumor xenografts in athymic mice were used to examine the effect of anti-Flk-1 mAb treatment on tumor angiogenesis and growth. Anti-Flk-1 mAb treatment significantly suppressed the growth of primary murine Lewis lung, 4T1 mammary, and B16 melanoma tumors and growth of Lewis lung metastases. DC101 also completely inhibited the growth of established epidermoid, glioblastoma, pancreatic, and renal human tumor xenografts. Histological examination of anti-Flk-1 mAb-treated tumors showed evidence of decreased microvessel density, tumor cell apoptosis, decreased tumor cell proliferation, and extensive tumor necrosis. These findings support the conclusion that anti-Flk-1 mAb treatment inhibits tumor growth by suppression of tumor-induced neovascularization and demonstrate the potential for therapeutic application of anti-VEGF receptor antibody in the treatment of angiogenesis-dependent tumors.

Identification and initial characterization of mSLK, a murine member of the STE20 family of kinases.

Protein kinases play a major role in the regulation of cellular growth. We isolated a murine cDNA encoding a novel serine/threonine kinase (referred to as mSLK) ubiquituously expressed during all stages of murine development and in all adult tissues examined. The cDNA codes for a 1233-amino-acid polypeptide characterized by an N-terminal catalytic domain and a large C-terminal region of unknown function. The sequence of the catalytic domain places mSLK in the STE20 family of cytoplasmic kinases. The noncatalytic domain does not show significant homology to any known protein. mSLK expressed in either COS7 cells or in bacteria was shown to contain kinase activity. The N-terminal fragment of mSLK was able to autophosphorylate on serine and threonine residues, phosphorylate threonine residues on a C-terminal fragment of the molecule, and phosphorylate exogenous substrates myelin basic protein and histone III in vitro. Furthermore, autophosphorylation of the catalytic domain was enhanced in the presence of the C-terminal fragment, which suggests a possible regulatory role for the C-terminal region of mSLK. A genomic clone of mSLK was isolated and used for fluorescence in situ hybridization locating the mSLK gene on band D2 of mouse chromosome 19.

Monoclonal antibodies targeting the VEGF receptor-2 (Flk1/KDR) as an anti-angiogenic therapeutic strategy.

Biological evidence suggests that interference with the function of the angiogenic growth factor receptor VEGFR2 (flk1/KDR) is a particularly promising strategy to inhibit tumor-induced angiogenesis. Proof of concept was established by developing a monoclonal rat anti-mouse VEGFR2 antibody (DC101) and showing that it potently blocked the binding of VEGF to its receptor, inhibited VEGF-induced signaling, and strongly blocked tumor growth in mice through an anti-angiogenic mechanism. Since DC101 does not cross-react with the human VEGFR2 KDR, anti-KDR monoclonal antibodies were generated by standard hybridoma technology and by using phage display library. High affinity antibodies (Kd = 4.9 x 10(-10)-1.1 x 10(-9) M) were found with both approaches. The anti-KDR antibodies compete on an equimolar basis with VEGF for binding to KDR and inhibit with similar potency the VEGF-induced signaling and mitogenesis in human endothelial cells. Although these antibodies cannot be tested for in vivo efficacy in standard murine tumor models because of lack of species cross-reactivity, the similarity of their in vitro properties with those of DC101 suggests that they may be effective in blocking KDR function in vivo.

Inhibition of vascular endothelial growth factor-induced receptor activation with anti-kinase insert domain-containing receptor single-chain antibodies from a phage display library.

A single-chain antibody phage display library was constructed from spleen cells of mice immunized with a soluble form of a human vascular endothelial growth factor (VEGF) receptor, kinase insert domain-containing receptor (KDR). After two rounds of biopanning, >90% of the clones recovered were specifically reactive to KDR. Subsequent selection identified two clones that blocked VEGF binding to KDR. The clones were expressed in Escherichia coli and purified as soluble single-chain Fv (scFv) antibodies. The affinities of the scFv for binding to KDR were determined by BIAcore analysis (2.1 x 10(-9)-5.9 x 10(-9) M). One scFv, p1C11, was shown to inhibit VEGF-induced KDR phosphorylation and VEGF-stimulated DNA synthesis in human umbilical vein endothelial cells. There is much experimental evidence to suggest that the VEGF/KDR/Flk-1 pathway plays an important role in tumor angiogenesis, a process that is essential for tumor growth and metastasis. The antibodies discussed here, which block VEGF binding to KDR, have potential clinical application in the treatment of cancer and other diseases where pathological angiogenesis is involved.

Hematopoietic activity of a stromal cell transmembrane protein containing epidermal growth factor-like repeat motifs.

Primitive hematopoietic stem cells are closely associated with discrete in vivo microenvironments. These "niches" are thought to provide the molecular signals that mediate stem cell differentiation and self-renewal. We have dissected the fetal liver microenvironment into distinct cellular components by establishing an extensive panel of stromal cell lines. One particular cell line maintains repopulating stem cells for prolonged in vitro culture periods. A subtraction cloning strategy has yielded a cDNA that encodes a cell surface glycoprotein with a restricted pattern of expression among stromal cell lines. This molecule, previously identified as delta-like/preadipocyte factor-1, contains epidermal growth factor-like repeats that are related to those in the notch/delta/serrate family of proteins. We have investigated the potential role of this molecule in hematopoietic stem/progenitor cell regulation. We show that the delta-like protein displays activity on purified stem cells by promoting the formation of "cobblestone areas" of proliferation. These cobblestone areas contain both primitive high-proliferative potential progenitors and in vivo repopulating stem cells.

The effects of Flk-2/flt3 ligand as compared with c-kit ligand on short-term and long-term proliferation of CD34+ hematopoietic progenitors elicited from human fetal liver, umbilical cord blood, bone marrow, and mobilized peripheral blood.

The Flk-2/flt3 ligand (FL) was evaluated and compared with c-kit ligand (KL) for its in vitro proliferative effects on CD34+ cells from human fetal liver, umbilical cord blood, bone marrow, and mobilized peripheral blood. Using a 7-day liquid culture system, FL in combination with interleukin-3 (IL-3), interleukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF) was comparable with KL in combination with IL-3, IL-6, and G-CSF for the expansion of hematopoietic progenitors. When FL-containing cultures were assayed after 21 or 28 days, a greater number of progenitors were generated as compared with KL-containing cultures. Using bone marrow microvascular endothelial cells as support stroma, cultures supplemented with FL generated a greater number of progenitors in both the nonadherent and adherent layers at day 35. These data suggest that FL ligand, in combination with other cytokines, can be used for short-term ex vivo expansion of hematopoietic progenitors and facilitates the preservation and possible expansion of primitive cells capable of long-term generation of progenitors.

Isolation and characterization of a monoclonal antibody binding to the extracellular domain of the flk-2 tyrosine kinase receptor.

The flk-2 tyrosine kinase receptor is expressed on hematopoietic stem cells and on acute leukemias (AML and ALL). We have isolated a rat monoclonal antibody (71E1) that binds to this receptor with a relative affinity of 5 nM. The antibody immunoprecipitates both murine and human forms of flk-2 and can block receptor activation by its cognate ligand. In addition, 71E1 inhibits the in vitro proliferation of the murine leukemic cell line, M1, that expresses high levels of flk-2. These results suggest that 71E1 may have utility as both a reagent for elucidating the biological role of flk-2 in hematopoiesis and as an immunotherapeutic in the treatment of acute leukemias.

An internalization motif is created in the cytoplasmic domain of the transferrin receptor by substitution of a tyrosine at the first position of a predicted tight turn.

Receptors are internalized from the plasma membrane at approximately 10 times the rate of bulk membrane. The predominant model for the motif that promotes rapid internalization proposes a requirement for a tyrosine located in the first position of a tight turn. In this report we show that an internalization motif can be created de novo by substituting a tyrosine for the first or last residues of a tetrapeptide GDNS (residues 31-34) that is predicted to form a tight turn within the cytoplasmic domain of the human transferrin receptor. These substitutions restore wild-type levels of internalization to transferrin receptors that are poorly internalized due to missense mutations in the native internalization motif. The introduction of a tyrosine at the first or last position of the GDNS tetrapeptide in a transferrin receptor containing an unmodified wild-type internalization motif significantly increases the internalization rate above that of the wild-type receptor. Our results indicate that a functional novel internalization motif can be created by placing specific aromatic amino acids within the overall structure of an existing beta-turn in a cytoplasmic domain of a receptor.

Endosome acidification and receptor trafficking: bafilomycin A1 slows receptor externalization by a mechanism involving the receptor's internalization motif.

To examine the relationship between endosome acidification and receptor trafficking, transferrin receptor trafficking was characterized in Chinese hamster ovary cells in which endosome acidification was blocked by treatment with the specific inhibitor of the vacuolar H(+)-ATPase, bafilomycin A1. Elevating endosome pH slowed the receptor externalization rate to approximately one-half of control but did not affect receptor internalization kinetics. The slowed receptor externalization required the receptor's cytoplasmic domain and was largely eliminated by substitutions replacing either of two aromatic amino acids within the receptor's cytoplasmic YTRF internalization motif. These results confirm, using a specific inhibitor of the vacuolar proton pump, that proper endosome acidification is necessary to maintain rapid recycling of intracellular receptors back to the plasma membrane. Moreover, receptor return to the plasma membrane is slowed in the absence of proper endosome acidification by a signal-dependent mechanism involving the receptor's cytoplasmic tyrosine-containing internalization motif. These results, in conjunction with results from other studies, suggest that the mechanism for clustering receptors in plasma membrane clathrin-coated pits may be an example of a more general mechanism that determines the dynamic distribution of membrane proteins among various compartments with luminal acidification playing a crucial role in this process.

Mutagenesis of the human transferrin receptor: two cytoplasmic phenylalanines are required for efficient internalization and a second-site mutation is capable of reverting an internalization-defective phenotype.

Site-specific mutagenesis has been used to define the sequences required for efficient internalization of the human transferrin receptor. It has previously been shown that the sole cytoplasmic tyrosine, at position 20, is required for efficient internalization. When two other cytoplasmic aromatic residues, the phenylalanines at positions 13 and 23, are substituted with alanines internalization is also reduced. The phenylalanine 23 mutation decreases the internalization rate constant approximately threefold, and mutation of phenylalanine 13 decreases it by approximately twofold. The mutation at position 23 has as serious an effect on internalization as substitution with a nonaromatic amino acid for the single tyrosine. These results demonstrate the importance of several aromatic amino acids in maintaining efficient internalization of the transferrin receptor. Substitution of a tyrosine at a second site, for a serine at position 34, within the cytoplasmic domain of a transferrin receptor with a nonaromatic amino acid at position 20, results in a complete reversion of the internalization-defective phenotype. This reversion is completely dependent upon a tyrosine, as phenylalanine substituted at position 34 does not revert the internalization-defective phenotype. This result demonstrates that a tyrosine placed outside of its native context can still function in the internalization of the transferrin receptor, suggesting a flexibility in surrounding sequences required for efficient internalization.

Fc and C3bi receptors and the differentiation antigen BH2-Ag are randomly distributed in the plasma membrane of locomoting neutrophils.

Reports from several laboratories suggest that neutrophils arrested during locomotion preferentially bind immune complexes at the front of the cell. Such asymmetry of binding has been interpreted as indicating an active modulation of phagocytic receptors to the anterior of the cell. To investigate this further, we have used digital analysis of fluorescence images to determine the binding patterns of mAbs directed against the Fc receptors, the receptors for the C3bi fragment of C3, and a neutrophil-specific antigen. We found that all three proteins are distributed nearly identically along the length of migrating neutrophils, and their distribution very closely parallels the anterior to posterior distribution of the plasma membrane. The use of mAbs offered an important advantage in that the binding of antireceptor antibodies, unlike the binding of ligands, should be independent of potential changes in the affinity of the receptors. We conclude that the anterior distribution of the phagocytic receptors in the plasma membrane of locomoting neutrophils parallels the overall increase in membrane area at the front of a migrating cell and that specific translocation of phagocytic receptors does not occur.

A monoclonal antibody to a human neutrophil-specific plasma membrane antigen. Effect of the antibody on the C3bi-mediated adherence by neutrophils and expression of the antigen during myelopoiesis.

We have used mice selectively tolerized to antigens of human lymphocytes by treatment with cyclophosphamide to raise an mAb, BH2-C6, that reacts with a plasma membrane antigen specific for human neutrophils. This specificity is demonstrated by indirect immunofluorescence microscopy, cytochemical analysis of fluorescence-positive and -negative cell populations separated by flow cytometry, and by the selective, complement-mediated killing of mAb BH2-C6-treated neutrophils. Additional evidence for the neutrophil specificity of mAb BH2-C6 is shown by immunoelectron microscopy, which demonstrates a lack of reactivity with human eosinophils. Immunoblotting of SDS-PAGE-separated proteins of polymorphonuclear leukocytes with 125I-labeled BH2-C6 identifies protein with an average molecular mass of 157 kD. Binding studies show that, at saturation, neutrophils bind 214,000 molecules of 125I-BH2-C6 per cell. Addition of mAb BH2-C6 to neutrophils significantly reduces the number of C3bi-opsonized sheep erythrocytes (EIgMC3bi) bound by these cells. This reduction is partly reversed by the presence of soybean trypsin inhibitor (SBTI), indicating that at least one part of this inhibition is due to BH2-C6-stimulated secretion of a serine protease that may affect ligand binding. Cytochemical analysis of normal human bone marrow cells sorted by cytofluorimetry identifies the promyelocyte as the precursor cell that first expresses BH2-Ag on the plasma membrane. Using the leukemic cell line HL-60, we demonstrate that only inducers of granulocytic differentiation, cis-retinoic acid, and dimethyloxazolidine stimulate the expression of BH2-Ag. These results show that the expression of BH2-Ag during myelomonocytic differentiation is a property uniquely possessed by cells committed to the neutrophilic lineage.