Biomedical subjects

S D Lyman

Publications and source records attributed to S D Lyman.

At least 73 records · Page 4Linked to original sources

PubMed1995

The flt3 ligand supports proliferation of lymphohematopoietic progenitors and early B-lymphoid progenitors.

We have examined the effects of the murine ligand (FL) for the flt3/flk2 tyrosine kinase receptor on the proliferation of murine lymphohematopoietic progenitors as well as committed myeloid and B-cell progenitors. In the presence of erythropoietin, FL alone supported scant colony formation from enriched marrow cells of normal mice. However, when it was combined with interleukin-3 (IL-3), steel factor (SF), or IL-11, FL significantly enhanced colony formation. When tested on enriched marrow cells from 5-fluorouracil (5-FU)-treated mice, FL neither enhanced IL-3-dependent colony formation nor synergized with SF in support of colony formation. However, FL synergized with IL-6, IL-11, or granulocyte-colony stimulating factor (G-CSF) in support of formation of various types of colonies, including multilineage colonies. Approximately 30% of these colonies yielded pre-B-cell colonies when replated in secondary cultures containing SF and IL-7, indicating that 2-cytokine combinations, including FL and IL-6, IL-11, or G-CSF can support the proliferation of primitive lymphohematopoietic progenitors. FL, by itself and in synergy with IL-7 or SF, supported the proliferation of B-cell progenitors. These results show that FL has a wide range of activities in early hematopoiesis and B lymphopoiesis.

Animals↗

PubMed1995

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).

Amino Acid Sequence↗

PubMed1995

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.

Alternative Splicing↗

PubMed1995

Isolation of LERK-5: a ligand of the eph-related receptor tyrosine kinases.

Hek and elk are members of the eph-related family of receptor tyrosine kinases. Recently we isolated four cDNAs encoding membrane-bound ligands to hek and elk [Beckman et al. (1994) EMBO J. 13, 3757-3762; Kozlosky et al. (1995) Oncogene 10, 299-306]. Because of the promiscuous nature of their binding, we have termed these proteins ligands of the eph-related kinases or LERKs. A search of GenBank revealed an expressed sequence tag (EST) with homology to the LERKs. Using this EST as a probe, we have isolated human and murine cDNAs that encode a protein which we call LERK-5. The human and murine cDNAs encode proteins of 333 and 336 amino acids, respectively, with a 97% amino acid identity; LERK-5 has an amino acid identity of 27-59% with the other reported LERKs. LERK-5 is a ligand for both elk and hek and induces receptor phosphorylation. It is expressed in adult lung and kidney and the fetal tissues heart, lung, kidney, and brain. In addition, Southern blot analysis of DNA from interspecific backcross mice indicated that LERK-5 (Eplg5) maps to the proximal region of mouse chromosome 8.

Amino Acid Sequence↗

PubMed1995

Biology of flt3 ligand and receptor.

The flt3 ligand is a member of a small family of growth factors that stimulate the proliferation of hematopoietic cells; other members of this family include Steel factor (also known as mast cell growth factor, stem cell factor, and kit ligand) and colony stimulating factor 1. These proteins function by binding to and activating unique tyrosine kinase receptors. Expression of the flt3 receptor is primarily restricted among hematopoietic cells to the most primitive progenitor cells. The flt3 ligand is similar to Steel factor in that both proteins stimulate the proliferation of early progenitor or stem cells. Neither of these factors has much proliferative activity on its own, but each factor can synergize with a wide range of other colony stimulating factors and interleukins (ILs) to stimulate proliferation. One major difference between the two factors appears to be their effect on mast cells, which Steel factor stimulates but flt3 ligand does not. Although flt3 ligand and Steel factor each act on early hematopoietic cells, differences in their activities suggest that they are not redundant and both are required for normal hematopoiesis. There are a number of clinical settings in which the flt3 ligand may potentially prove quite useful.

Amino Acid Sequence↗

PubMed1995

Biology and potential clinical applications of flt3 ligand.

The flt3 ligand is a member of a small family of growth factors that stimulate the proliferation of hematopoietic cells. Other members of this family include Steel factor (also known as mast cell growth factor, stem cell factor, and kit ligand) and colony-stimulating factor 1. These proteins function by binding to and activating unique tyrosine kinase receptors. Both flt3 ligand and Steel factor stimulate the proliferation of early progenitor or stem cells. Neither of these factors exhibits much biologic activity by itself, but each factor can synergize with a wide range of other colony-stimulating factors and interleukins. One major difference between the two factors appears to be their effect on mast cells, which Steel factor stimulates, but flt3 ligand does not. Although flt3 ligand and Steel factor each act on early hematopoietic cells, differences in their activities suggest that they are not redundant and are both required for normal hematopoiesis. There are a number of clinical settings in which the flt3 ligand may prove quite useful.

Animals↗

PubMed1995

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.

Amino Acid Sequence↗

PubMed1995

Flt3 ligand stimulates/costimulates the growth of myeloid stem/progenitor cells.

The present studies evaluated effects of recombinant human (rhu) and murine (rmu) flt3 ligand (flt3-L) on colony formation by subsets of myeloid stem and progenitor cells present in low-density (LD) and cell-sorted CD34 hu cord blood (CB) and bone marrow (BM) cells and unseparated mu BM cells. By itself, flt3-L had weak colony-stimulating activity. It stimulated small dispersed CFU-GM-type colonies, but not BFU-E, CFU-GEMM, or HPP-CFC colonies, from LD and CD34 huCB and BM. However, flt3-L had additive to greater-than-additive effects on colony number and size by CFU-GM stimulated with GM-CSF or IL-3, with or without Steel factor (SLF); by CFU-G stimulated by G-CSF with or without SLF; by CFU-M stimulated by CSF-1; and by BFU-E, CFU-GEMM, and HPP-CFC stimulated by Epo with or without IL-3 or SLF. Flt3-L enhanced the effects of SLF, alone and in combination with other CSFs. Similar effects were apparent on LD and sorted CD34 cells and also at the level of single sorted and isolated CD34 cells/well. Flt3-L enhanced expansion of immature subsets of huCD34(+)-column separated CB CFU-GM stimulated by the potent combination of SLF and PIXY321 (a GM-CSF/IL-3 fusion protein). While flt3-L did not enhance the replating capacity of CFU-GEMM plated in the presence of Epo and SLF, it enhanced numbers of these CFU-GEMM colonies with the capacity to be replated. Flt3-L effects were not species-specific; rhu and rmu forms were active on huCB/BM and muBM. These results demonstrate the potent direct-acting stimulating/costimulating activities of flt3-L in vitro on myeloid stem/progenitor cells.

Adult↗

PubMed1995

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.

Animals↗

PubMed1994

Genomic organization and chromosomal localization of mouse Eplg2, a gene encoding a binding protein for the receptor tyrosine kinase elk.

The human gene EPLG2 (Eph ligand-2) encodes a potential ligand for the receptor tyrosine kinase elk. High sequence conservation between the human and the rat cDNAs and developmentally regulated expression of the rat gene suggest that the protein encoded by EPLG2 plays an important role in mammalian development. To facilitate analysis of the physiological role of the protein, we have cloned and characterized a 24-kb region of mouse genomic DNA containing the mouse homologue of EPLG2 (Eplg2), including 5'- and 3'-flanking sequences. Restriction mapping, coupled with Southern blot hybridization and sequencing, was used to determine the structural organization of the gene. The Eplg2 genomic locus spans a region of approximately 12 kb, encoding five exons and four introns. The first intron comprises approximately 8.5 kb of the entire 12-kb genomic sequence. Eplg2 was mapped to the mouse X chromosome by interspecific backcross analysis and is tightly linked to the androgen receptor (Ar) locus.

Amino Acid Sequence↗

PubMed1994

Cloning of the human homologue of the murine flt3 ligand: a growth factor for early hematopoietic progenitor cells.

Using a fragment of the murine flt3 ligand as a probe, we have succeeded in cloning a human flt3 ligand from a human T-cell lambda gt10 cDNA library. The human and murine ligands are 72% identical at the amino acid level. Analysis of multiple cDNA clones shows that alternative splicing of the human flt3 mRNA can occur at a number of positions. A recombinant soluble form of the human flt3 ligand stimulates the proliferation and colony formation of a subpopulation of human bone marrow cells that are CD34+ and are enriched for primitive hematopoietic cells. In addition, the human flt3 ligand also stimulates the proliferation of cells expressing murine flt3 receptors. Northern blot analysis shows widespread expression of flt3 ligand mRNA transcripts in human tissues.

Amino Acid Sequence↗

PubMed1994

The flt3 ligand: a hematopoietic stem cell factor whose activities are distinct from steel factor.

A number of growth factors have been described that affect the hematopoietic system. Among this group are Steel factor (also known as mast cell growth factor, stem cell factor and kit ligand), and the more recently described flt3 ligand. These factors have been shown to function by binding to and activating the c-kit and flt3 tyrosine kinase receptors, respectively. Both of these factors stimulate the growth of mouse and human hematopoietic progenitor cells. These factors therefore differ from such later acting hematopoietic factors as colony-stimulating factor (CSF)-1, which regulates the growth, survival and differentiation of monocytic cells through the c-fms tyrosine kinase receptor. Like Steel factor, the flt3 ligand has little biological activity on its own, but synergizes well with a number of other colony stimulating factors and interleukins. One major difference between the two factors appears to be their effect on mast cells. Steel factor stimulates both the proliferation and activation of mast cells, while preliminary data with the flt3 ligand suggests that it has no effect on mast cells. Although the flt3 ligand and Steel factor each act on early hematopoietic cells, differences in their activities suggest that they are not redundant and are both required for normal hematopoiesis.

Animals↗

PubMed1993

Molecular cloning of a ligand for the flt3/flk-2 tyrosine kinase receptor: a proliferative factor for primitive hematopoietic cells.

Cloning of a ligand for the murine flt3/flk-2 tyrosine kinase receptor was undertaken using a soluble form of the receptor to identify a source of ligand. A murine T cell line, P7B-0.3A4, was identified that appeared to express a cell surface ligand for this receptor. A cDNA clone was isolated from an expression library prepared from these cells that was capable, when transfected into cells, of conferring binding to a soluble form of the flt3/flk-2 receptor. The cDNA for this ligand encodes a type I transmembrane protein that stimulates the proliferation of cells transfected with the flt3/flk-2 receptor. A soluble form of the ligand stimulates the proliferation of defined subpopulations of murine bone marrow and fetal liver cells as well as human bone marrow cells that are highly enriched for hematopoietic stem cells and primitive uncommitted progenitor cells.

Amino Acid Sequence↗

PubMed1993

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.

Amino Acid Sequence↗

PubMed1993

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.

Amino Acid Sequence↗

PubMed1992

Enhancement of murine hematopoiesis by synergistic interactions between steel factor (ligand for c-kit), interleukin-11, and other early acting factors in culture.

Entry into the cell cycle of dormant hematopoietic progenitors appears to be regulated by multiple synergistic factors, including interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), IL-11, and the ligand for c-kit, which is also known as steel factor (SF). We have tested the effects of these and other hematopoietic factors on the proliferation of partially enriched dormant murine progenitors in the presence and absence of serum. In serum-containing cultures, SF and IL-11 interacted to support the formation of multilineage colonies; the level of colony formation was comparable with the colony formation supported by other effective two-factor combinations. In serum-free cultures, colony formation supported by two factors was significantly less than that in serum-containing culture and the most effective two-factor combination in serum-free culture was SF plus IL-3. In serum-free cultures, three-factor combinations consisting of SF, IL-3, and one of IL-6, G-CSF, or IL-11 yielded colony formation that was comparable with that seen in serum-containing cultures. These studies indicate that IL-11 belongs to a group of early-acting hematopoietic synergistic factors that now includes IL-6, G-CSF, and IL-11. In contrast, SF is unique among the synergistic factors in that it interacts either with growth factors such as IL-3 or GM-CSF or with synergistic factors such as IL-6, IL-11, or G-CSF.

Animals↗

PubMed1992

Influence of steel factor on hemoglobin synthesis in sickle cell disease.

A new hematopoietic growth factor (Steel factor) has been identified which stimulates erythroid proliferation both in vitro and in vivo. We evaluated the influence of recombinant Steel factor on hemoglobin synthesis in peripheral blood (PB) BFU-E-derived cells from normal donors by radioimmunoassay (RIA) and compared it with stimulation with GM-CSF and interleukin-3 (IL-3). Only Steel factor stimulated a significant increase in BFU-E-derived colony size and a significant increase in fetal hemoglobin (HbF) in BFU-E-derived erythroblasts from 0.49% +/- 0.27% to 6.33% +/- 1.11% in serum-deprived media and from 1.88% +/- 0.24% to 11.17% +/- 0.91% in serum. To determine whether this influence on hemoglobinization also occurred in sickle cell disease, we studied 13 patients with sickle cell disease. In serum-deprived conditions, there was a significant increase in the number and size of BFU-E-derived colonies with Steel factor that was dose-dependent. In addition, the proportion of HbF in progenitor-derived cells increased by 66% from 4.1% +/- 0.6% to 6.8% +/- 1.2% with Steel factor. In serum-containing conditions studied in 12 patients, the increase in percentage of HbF was even greater, from 10.7% +/- 0.9% in control cultures to 22.5% +/- 2.6% with Steel factor. These increases in percentage of HbF were significant and dose-dependent. An increase in percentage of HbF was observed in erythroblasts harvested on day 11, 14, and 18 of culture. A decrease in mean picograms of total Hb per cell after coculture with Steel factor was noted, suggesting that growth kinetics influenced complete hemoglobinization. In serum-deprived conditions, picograms of HbF per cell was not affected by Steel factor, and in serum-containing conditions that augment in vitro HbF production it was enhanced. Thus, Steel factor stimulated a significant increase in percentage of HbF in erythroid cells from normal donors and patients with SCA in vitro.

Anemia, Sickle Cell↗

PubMed1992

Recombinant murine steel factor stimulates in vitro production of granulocyte-macrophage progenitor cells.

The ability of murine Steel factor to promote the in vitro production of granulocyte-macrophage progenitor cells (CFU-GM) was examined in short-term liquid cultures. Bone marrow from C57BL/6J or Sl/Sld mice was placed in culture for seven days with either Steel factor alone or in the presence of IL-3. CFU-GM responsive to GM-CSF, IL-3, and CSF-1 were measured in the input population and again after 3 or 7 days in culture. Steel factor alone increased the number of all CFU-GM types as early as 3 days after culture initiation, with further increases at day 7. This effect was potentiated by the addition of IL-3. Production of CFU-GM by C57BL/6J or Sl/Sld marrow was comparable except for enhanced production of CSF-1 responsive progenitors by Sl/Sld marrow. A recombinant Sld protein was also shown to be equivalent to the wild-type protein in its capacity to promote CFU-GM production from normal bone marrow.

Animals↗