The FLT3 ligand potently and directly stimulates the growth and expansion of primitive murine bone marrow progenitor cells in vitro: synergistic interactions with interleukin (IL) 11, IL-12, and other hematopoietic growth factors.

The recently cloned murine flt3 ligand (FL) was studied for its ability to stimulate the growth of primitive (Lin-Sca-1+) and more committed (Lin-Sca-1-) murine bone marrow progenitor cells, alone and in combination with other hematopoietic growth factors (HGFs). Whereas FL was a weak proliferative stimulator alone, it potently synergized with a number of other HGFs, including all four colony-stimulating factor (CSF), interleukin (IL) 6, IL-11, IL-12, and stem cell factor (SCF), to promote the colony formation of Lin-Sca-1+, but not Lin-Sca-1- or erythroid progenitor cells. The synergistic activity of FL was concentration dependent, with maximum stimulation occurring at 250 ng/ml, and was observed when cells were plated at a concentration of one cell per culture, suggesting that its effects are directly mediated. 2 wk of treatment with FL in combination with IL-3 or SCF resulted in the production of a high proportion of mature myeloid cells (granulocytes and macrophages), whereas the combination of FL with G-CSF, IL-11, or IL-12 resulted predominantly in the formation of cells with an immature blast cell appearance. Accordingly, FL in combination with G-CSF or IL-11 expanded the number of progenitors more than 40-fold after 2 wk incubation. Thus, FL emerges as a potent synergistic HGF, that in combination with numerous other HGFs, can directly stimulate the proliferation, myeloid differentiation, and expansion of primitive hematopoietic progenitor cells.

Dramatic increase in the numbers of functionally mature dendritic cells in Flt3 ligand-treated mice: multiple dendritic cell subpopulations identified.

Dendritic cells (DC) are the most efficient APC for T cells. The clinical use of DC as vectors for anti-tumor and infectious disease immunotherapy has been limited by their trace levels and accessibility in normal tissue and terminal state of differentiation. In the present study, daily injection of human Flt3 ligand (Flt3L) into mice results in a dramatic numerical increase in cells co-expressing the characteristic DC markers-class II MHC, CD11c, DEC205, and CD86. In contrast, in mice treated with either GM-CSF, GM-CSF plus IL-4, c-kit ligand (c-kitL), or G-CSF, class II+ CD11c+ cells were not significantly increased. Five distinct DC subpopulations were identified in the spleen of Flt3L-treated mice using CD8 alpha and CD11b expression. These cells exhibited veiled and dendritic processes and were as efficient as rare, mature DC isolated from the spleens of untreated mice at presenting allo-Ag or soluble Ag to T cells, or in priming an Ag-specific T cell response in vivo. Dramatic numerical increases in DC were detected in the bone marrow, gastro-intestinal lymphoid tissue (GALT), liver, lymph nodes, lung, peripheral blood, peritoneal cavity, spleen, and thymus. These results suggest that Flt3L could be used to expand the numbers of functionally mature DC in vivo for use in clinical immunotherapy.

Human interleukin 4 receptor confers biological responsiveness and defines a novel receptor superfamily.

IL-4, a pleiotropic cytokine produced by T lymphocytes, plays an important role in immune responsiveness by regulating proliferation and differentiation of a variety of lymphoid and myeloid cells via binding to high affinity receptors. In this report we describe the isolation and functional expression of a human IL-4-R cDNA. When transfected into COS-7 cells, the cDNA encodes a 140-kD cell-surface protein. After transfection into a murine T cell line, the cDNA encodes a protein that binds human IL-4 with high affinity and can confer responsiveness to human IL-4. The predicted extracellular domain of the IL-4-R exhibits significant amino acid sequence homology with the beta subunit of the IL-2-R (p75), and the receptors for IL-6, erythropoietin, and prolactin. These receptors comprise a novel superfamily with extracellular domains characterized by four conserved cysteine residues and a double tryptophan-serine (WSXWS) motif located proximal to the transmembrane region.

A new cytokine receptor superfamily.

The amino acid sequences of several, recently cloned cytokine receptors show significant homologies, primarily in their extracellular, ligand-binding domains. With one exception, their cognate cytokines mediate biological activities on a variety of hematopoietic cell types; thus we have designated the receptors as the hematopoietic receptor superfamily.

c-KIT expression enhances the leukemogenic potential of 32D cells.

The growth of human leukemic cells in culture and in vivo is dependent upon the presence of hematopoietic growth factors. Most populations of human leukemic acute myeloblastic leukemia (AML) cells express c-Kit on their surface and respond to Kit ligand (KL) in culture. To determine if this interaction was of potential significance in vivo we used a mouse model system. 32D cells, a murine IL-3-dependent myeloid cell line, were rendered KL responsive by transfection of the murine c-Kit. After injection of 32D or 32D-Kit cells into syngeneic hosts, animals bearing 32D-Kit cells, but not 32D cells, became moribund and were killed. These animals had circulating leukemic blast cells, infiltration of bone marrow, spleen, brain, liver, lung, and kidney. Cells recovered from some of the animals continued to be dependent upon IL-3 or KL for growth while in other cases the cells were factor independent. This model illustrates that the constitutive expression of c-Kit enhances the leukemic potential of 32D cells. The model will be useful for studying the progression of leukemia in vivo and testing whether interruption of the interaction of Kit and KL can affect the growth of leukemic cells.

Analysis of functional domains of the v-fms-encoded protein of Susan McDonough strain feline sarcoma virus by linker insertion mutagenesis.

The Susan McDonough strain of feline sarcoma virus contains an oncogene, v-fms, which is capable of transforming fibroblasts in vitro. The mature protein product of the v-fms gene (gp140fms) is found on the surface of transformed cells; this glycoprotein has external, transmembrane, and cytoplasmic domains. To assess the functional role of these domains in transformation, we constructed a series of nine linker insertion mutations throughout the v-fms gene by using a dodecameric BamHI linker. The biological effects of these mutations on the function and intracellular localization of v-fms-encoded proteins were determined by transfecting the mutated DNA into Rat-2 cells. Most of the mutations within the external domain of the v-fms-encoded protein eliminated focus formation on Rat-2 cells; three of these mutations interfered with the glycosylation of the v-fms protein and interfered with formation of the mature gp140fms. One mutation in the external domain led to cell surface expression of v-fms protein even in the absence of complete glycosylational processing. Cell surface expression of mutated v-fms protein is probably necessary, but is not sufficient, for cell transformation since mutant v-fms protein was found on the surface of several nontransformed cell lines. Mutations that were introduced within the external domain had little effect on in vitro kinase activity, whereas mutations within the cytoplasmic domain all had strong inhibitory effects on this activity.

The Steel/W transduction pathway: kit autophosphorylation and its association with a unique subset of cytoplasmic signaling proteins is induced by the Steel factor.

The W/c-kit and Steel loci respectively encode a receptor tyrosine kinase (Kit) and its extracellular ligand, Steel factor, which are essential for the development of hematopoietic, melanocyte, and germ cell lineages in the mouse. To determine the biochemical basis of the Steel/W developmental pathway, we have investigated the response of the Kit tyrosine kinase and several potential cytoplasmic targets to stimulation with Steel in mast cells derived from normal and mutant W mice. In normal mast cells, Steel induces Kit to autophosphorylate on tyrosine and bind to phosphatidylinositol 3'-kinase (PI3K) and phospholipase C-gamma 1 but not detectably to Ras GTPase-activating protein. Additionally, we present evidence that Kit tyrosine phosphorylation acts as a switch to promote complex formation with PI3K. In mast cells from mice homozygous for the W42 mutant allele, Kit is not tyrosine phosphorylated and fails to bind PI3K following Steel stimulation. In contrast, in the transformed mast cell line P815, Kit is constitutively phosphorylated and binds to PI3K in the absence of ligand. These results suggest that Kit autophosphorylation and its physical association with a unique subset of cytoplasmic signaling proteins are critical for mammalian development.

c-Kit-kinase induces a cascade of protein tyrosine phosphorylation in normal human melanocytes in response to mast cell growth factor and stimulates mitogen-activated protein kinase but is down-regulated in melanomas.

The proto-oncogene c-Kit, a transmembrane receptor tyrosine kinase, is an important regulator of cell growth whose constitutively active oncogenic counterpart, v-kit, induces sarcomas in cats. Mutations in murine c-kit that reduce the receptor tyrosine kinase activity cause deficiencies in the migration and proliferation of melanoblasts, hematopoietic stem cells, and primordial germ cells. We therefore investigated whether c-Kit regulates normal human melanocyte proliferation and plays a role in melanomas. We show that normal human melanocytes respond to mast cell growth factor (MGF), the Kit-ligand that stimulates phosphorylation of tyrosyl residues in c-Kit and induces sequential phosphorylation of tyrosyl residues in several other proteins. One of the phosphorylated intermediates in the signal transduction pathway was identified as an early response kinase (mitogen-activated protein [MAP] kinase). Dephosphorylation of a prominent 180-kDa protein suggests that MGF also activates a phosphotyrosine phosphatase. In contrast, MGF did not induce proliferation, the cascade of protein phosphorylations, or MAP kinase activation in the majority of cells cultured from primary nodular and metastatic melanomas that grow independently of exogenous factors. In the five out of eight human melanoma lines expressing c-kit mRNAs, c-Kit was not constitutively activated. Therefore, although c-Kit-kinase is a potent growth regulator of normal human melanocytes, its activity is not positively associated with malignant transformation.

Colony stimulating factor-1 induced growth stimulation of v-fms transformed fibroblasts.

The v-fms oncogene, which is capable of transforming fibroblasts, was derived by recombination of a feline leukemia virus with a cellular gene (c-fms) that encodes the receptor for colony stimulating factor 1 (CSF-1). We examined the capacity of recombinant human CSF-1 (produced in a yeast expression system) to stimulate the growth of v-fms transformed rat fibroblasts. Recombinant human CSF-1 bound to v-fms transformed fibroblasts with high affinity (apparent Kd = 6.0 x 10(-10) M); only non-specific binding was observed on control cells. The number of colonies formed in soft agar by v-fms transformed cells was increased by CSF-1 treatment in a dose-dependent manner; a nine fold increase in the number of colonies was seen in the presence of 10(-8) M CSF-1. CSF-1 did not stimulate the growth of either non-transformed cell lines, a non-transformed cell line that expresses a mutated v-fms protein on the cell surface, or cells transformed by the v-fgr oncogene. The growth stimulating effect of CSF-1 on v-fms transformed cells was also seen in monolayer culture. The v-fms transformed cells treated with CSF-1 had a more refractile, rounded morphology than non-treated cells; no morphology change was observed in CSF-1 treated control cells. CSF-1 treatment also increased both the number and size of foci that arose from fibroblasts following transfection with the v-fms oncogene. These data show that the altered CSF-1 receptor encoded by the v-fms oncogene retains a capacity to bind, and be stimulated by, human CSF-1.

KIT ligand (mast cell growth factor) inhibits the growth of KIT-expressing melanoma cells.

Previous studies in vivo and in vitro show that KIT kinase promotes normal melanocyte development and growth. However, the role of the KIT proto-oncogene in neoplastic melanocytes is not certain. We therefore examined KIT expression and function in human melanomas. Our results show that KIT mRNA was expressed in 12 of 28 melanoma cell lines (approximately 40%), mainly in those originating from pigmented tumors. Surprisingly, activation of KIT with mast cell growth factor (MGF) in melanoma cells produced biological responses opposite to those elicited in normal melanocytes. MGF inhibited rather than stimulated the growth of metastatic melanoma cell lines. The opposite effects may be due to aberrant signal transduction by KIT in melanoma cells in response to MGF. The in vitro inhibition of melanoma cells by MGF suggests that growth in vivo of this tumor is not promoted by KIT kinase activation, but rather that transformed melanocytes might regress when MGF is expressed in their immediate environment.

Characterization of the protein encoded by the flt3 (flk2) receptor-like tyrosine kinase gene.

We have developed rabbit polyclonal antibodies to the C-terminus of the flt3-encoded protein, which is a member of the receptor tyrosine kinase family. Immunoprecipitation using this antiserum brings down two protein bands, a major band of 143 kDa and a less abundant, more diffuse, band of 158 kDa. Pulse-chase analysis of flt3 protein from transfected COS-7 cells shows that the larger band is derived from the smaller one and presumably represents maturation of the protein from a glycosylated high-mannose form to a complex carbohydrate form. N-glycosidase F digestion confirmed the presence of N-linked carbohydrates, and cell-surface labeling of flt3-transfected cells indicated that the 158-kDa glycoprotein is the species found on the cell surface. A mutated form of the flt3 protein that was defective in its glycosylational processing was identified. Western blotting of the immunoprecipitated flt3 protein showed that it is heavily phosphorylated on tyrosine, and that this phosphorylation probably occurs in the absence of ligand. In this regard, the flt3 protein resembles the c-erbB2 protein, which is also highly phosphorylated in the absence of ligand. These data suggest that the flt3 receptor regulates the growth and differentiation of cells via an as yet unknown ligand.

Structural analysis of human and murine flt3 ligand genomic loci.

Both the murine and human genomic loci that encode flt3 ligand have been cloned. flt3 ligand is a hematopoietic growth factor that stimulates the proliferation of stem and progenitor cells. The portions of the murine and human flt3 ligand genomic loci encompassing the coding region of the protein are approximately 4.0 kb and 5.9 kb, respectively. The human genomic locus is larger as a result of the presence of repeated sequences within introns I, II, IV, V and VI. The transmembrane isoform of the murine and human flt3 ligand proteins are each encoded within seven exons (1-5 + 7 and 8). Analyses of flt3 ligand cDNA clones show that alternative splicing of a putative sixth exon results in the generation of a soluble form of the flt3 ligand protein. The sizes of each of the exons are well conserved between species. Murine and human flt3 genomic loci have a similar exon: intron structure compared to the genomic loci encoding Steel factor and colony stimulating factor 1. These proteins, which appear to be ancestrally related, are hematopoietic growth factors that stimulate cells via specific and structurally related tyrosine kinase receptors on the cell surface.

Enforced c-KIT expression renders highly metastatic human melanoma cells susceptible to stem cell factor-induced apoptosis and inhibits their tumorigenic and metastatic potential.

Expression of the tyrosine-kinase receptor encoded by the c-KIT proto-oncogene progressively decreases during local tumor growth and invasion of human melanomas. To provide direct evidence that c-KIT plays a role in metastasis of human melanoma, we transfected the c-KIT gene into the c-KIT negative highly metastatic human melanoma cell line A375SM and subsequently analysed its tumorigenic and metastatic potential. A375SM parental cells, A375SM-NOT (neo, control), and A375SM-KIT-positive cells were injected s.c. and i.v. into nude mice. A375SM-KIT cells produced significantly slower growing s.c. tumors and fewer lung metastases than control cells. Exposure of c-KIT-positive melanoma cells in vitro and in vivo to stem cell factor (SCF), the ligand for c-KIT, triggered apoptosis of these cells but not of c-KIT-negative melanoma cells or normal melanocytes. Since SCF is produced by keratinocytes and other dermal cells in the skin, these results suggest that the loss of c-KIT receptor expression may allow malignant melanoma cells to escape SCF/c-KIT-mediated apoptosis, hence contributing to tumor growth and eventually metastasis. The antitumor and antimetastatic properties of SCF may be useful in treating human melanomas in early stages.

Ligands for the receptor tyrosine kinases hek and elk: isolation of cDNAs encoding a family of proteins.

Hek is a member of the eph subfamily of receptor tyrosine kinases whose members include elk, hek2, sek, eph and eck among others. Using a soluble form of hek consisting of the extracellular region of the receptor fused to the Fc domain of human IgG1 and an expression cloning strategy, we have isolated two different but related cDNAs from the human T-lymphoma line HSB-2 that encode ligands for hek. The cDNAs encode proteins of 238 and 201 amino acids (44% amino acid identity) that are anchored to the membrane by glycosylphosphatidylinositol (GPI)-linkage. The proteins encoded by these cDNAs are bound by hek with affinity constants of 2 x 10(8) M-1. These proteins also bind the elk tyrosine kinase receptor. These cDNAs are related to other cDNAs that we have recently isolated from a human placental library that encode ligands for both hek and elk and define an emerging family of ligands for eph-related kinases (LERKs).

Identification of soluble and membrane-bound isoforms of the murine flt3 ligand generated by alternative splicing of mRNAs.

We have recently described a novel hematopoietic growth factor, referred to as the flt3 ligand, that stimulates the proliferation of sub-populations of hematopoietic cells that are enriched for stem and progenitor cells. This factor is a transmembrane protein that undergoes proteolytic cleavage to generate a soluble form of the protein. We have isolated additional flt3 ligand isoforms by PCR that contain an extra exon and encode what are predicted to be either a soluble form of the ligand or a longer version of the transmembrane protein. We have also isolated cDNAs from murine T cell libraries that encode an isoform of the flt3 ligand that has an unusual C-terminus. This isoform results from a failure to splice out an intron during mRNA processing. The protein encoded by this cDNA is expressed on the cell surface, where it is biologically active. However, this novel isoform does not appear to give rise to a soluble form of the protein. Regulation of mRNA splicing is likely to control the generation of cell bound or soluble forms of this hematopoietic growth factor. Genetic mapping studies localize the gene encoding the flt3 ligand to the proximal portion of mouse chromosome 7 and to human chromosome 19q13.3.

Expression of the flt3 receptor and its ligand on hematopoietic cells.

Expression of the flt3 tyrosine kinase receptor and its ligand were examined on various murine and human hematopoietic cell lines. Surface expression of flt3 receptor and flt3 ligand were detected by flow cytometry using biotinylated human flt3 ligand or biotinylated soluble human flt3 receptor Fc fusion protein (flt3R-Fc), respectively. Flt3 receptor and ligand expression were also examined by Northern blot analysis. Flt3 receptor was expressed on the surface of only two of nine murine cell lines and nine of 15 human cell lines, with positive cells representing the B cell, early myeloid, and monocytic lineages. Staining for surface expression of the flt3 ligand revealed that seven of nine murine cell lines and nine of 15 human cell lines screened were positive by flow cytometry. All murine and human cell lines assayed were positive for flt3 ligand RNA expression by Northern blot analysis, but not all cell lines expressing flt3 ligand mRNA had detectable surface expression. Cells expressing the flt3 ligand were of the myeloid, B cell and T cell lineages at various stages of differentiation. Only the OCI-AML-5, NALM-6, and AML-193 cell lines coexpressed both surface flt3 receptor and ligand. The myeloid leukemic M1 cell terminally differentiate into macrophage-like cells under the influence of leukemia inhibitory factor (LIF). We found that LIF-stimulated M1 cells down-regulated surface expression and mRNA levels of the flt3 receptor, but up-regulated expression of the flt3 ligand. Although we could demonstrate that the flt3 receptor was functional in the M1 cell line, flt3 ligand could not induce the M1 cells to differentiate.

Endogenous FLT-3 ligand serum levels are associated with disease stage in patients with myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) caused by a clonal hematopoietic stem cell disorder progress to either overt leukemia or cytopenia, which leads to lethal infection or bleeding. Although several clinical trials have attempted to reverse cytopenia by using hematopoietic growth factors (HGF), success has been limited due in part to a limited understanding of the role of HGF in MDS progression. The FLT3 ligand, which binds to and activates the FLT3 receptor, does not have a stimulatory effect on hematopoietic cells, but can synergize with other HGF to support the expansion of both immature and committed progenitors. Using ELISA technology we measured endogenous serum levels in 93 patients with MDS: 29 RA, 1 RARS, 31 RAEB, 23 RAEBt, 9 CMML. 48.3% of RA patients' sera had significantly elevated FLT3 ligand levels ranging from 404 to 5735 pg/ml, whereas none of the RAEB, RAEBt, or CMML patients sera had levels different from controls. No significant correlation was found between FLT3 ligand levels and peripheral blood counts, bone marrow cellularity, age, cytogenetic abnormalities, or survival. Our data suggest that FLT3 ligand levels can be upregulated early in the course of MDS, which may represent an appropriate response to a decreased number of normal progenitors, or alternatively a dysregulated HGF system.

Thrombopoietin augments ex vivo expansion of human cord blood-derived hematopoietic progenitors in combination with stem cell factor and flt3 ligand.

We studied the effects of stem cell factor (SCF) and flt3 ligand (FL) on the ex vivo expansion of human umbilical cord blood (CB)-derived CD34+ cells in combination with various cytokines, including interleukin (IL)-3, IL-6, IL-11, and c-Mpl ligand (thrombopoietin, TPO), in a short-term serum-free liquid suspension culture system. Among the two-factor combinations tested, SCF plus IL-3 most effectively expanded committed progenitor cells, including mixed colony-forming units (CFU-Mix). The expansion efficiency (EE) of FL for each progenitor was inferior to that of SCF in the presence of various cytokines, except TPO. IL-6 significantly increased the EE for granulocyte/macrophage colony-forming units (CFU-GM) obtained with SCF + IL-3 or FL + IL-3. Interestingly, TPO markedly augmented the EE for committed progenitors, including CFU-GM, erythroid burst-forming units (BFU-E), and CFU-Mix, in the presence of SCF + IL-3 or FL + IL-3. The combinations of SCF + IL-3 + TPO + IL-6 or IL-11 maximally stimulated the expansion of committed progenitors. The maximum EE for CFU-GM, BFU-E, and CFU-Mix was respectively 197-fold (day 14), 60-fold (day 7) and 51-fold (day 14). Other combinations of cytokines without IL-3 failed to expand effectively these committed progenitors. Our data demonstrate that it is possible to expand human CB-derived committed progenitors in vitro using SCF or FL with several other cytokines including TPO, and that IL-3 is the key cytokine promoting the expansion of human hematopoietic progenitors in the presence of SCF or FL.

Flt3-ligand production by human bone marrow stromal cells.

Bone marrow stromal cells are important sources of cytokines and growth factors which participate in regulation of proliferation and differentiation of hematopoietic stem and progenitor cells. Recently flt3/flk-2-ligand (flt3-L), a new growth factor which uses a membrane tyrosine kinase receptor, was cloned. It is expressed in transmembrane and soluble forms and stimulates/co-stimulates proliferation and colony formation of hematopoietic stem/progenitor cells. It has not been reported whether flt3-L is produced by cells of the hematopoietic bone marrow microenvironment. We demonstrate the expression of flt3-L in bone marrow fibroblasts (BMF) and in stromal cells of adherent layers of long-term bone marrow cultures by RT-PCR, Northern blot, immunocytochemistry and FACS analysis. The latter two methods localized flt3-L intracellularly and on cell membranes. Treatment with interleukin-1 alpha increased the expression of flt3-L in BMF. This demonstrates production and modulation of flt3-L in stromal cells of human bone marrow.

Mast cell growth factor, a ligand for the receptor encoded by c-kit, affects the growth in culture of the blast cells of acute myeloblastic leukemia.

The c-kit proto-oncogene encodes a transmembrane receptor with a tyrosine kinase internal domain. C-kit has been mapped to the W locus in the mouse, and the gene encoding the ligand has been shown to be the product of the murine SI locus. Previous genetic studies have shown that the murine W and SI loci play important roles in the normal function of hemopoietic stem cells. As these stem cells have been identified as the origins of abnormal clones in acute myeloblastic leukemia (AML), a study was begun of c-kit in AML. By Northern blot analysis, it was shown that all of 21 blast populations from AML patients were kit expression positive, but some AML cell lines did not transcribe detectable c-kit mRNA. This study is now extended to the responses of freshly obtained AML cells and cell lines to the ligand, mast-cell growth factor (MGF). In culture, fresh cells usually responded to added ligand with increases in both self-renewal and terminal divisions. The most obvious effects were seen when MGF was combined with either IL-3 or G-CSF. The response of cell lines to MGF mirrored their expression of c-kit; expression positive lines responded in culture with patterns similar to those seen for fresh cells. C-kit expression negative cells did not respond to MGF. RNA prepared from the cells giving rise to one such line, OCI/AML-5, was available for study. mRNA for c-kit could not be detected in this RNA sample by Northern blot analysis or the polymerase chain reaction. Thus the heterogeneity found in AML blast populations extends to the involvement of c-kit and its ligand in growth regulation, although blast populations without this regulatory apparatus appear to be rare.