Evidence that stem cell factor is involved in the rebound thrombocytosis that follows 5-fluorouracil treatment.

The mechanisms responsible for 5-fluorouracil (5FU)-induced rebound thrombocytosis are not completely understood. SI/SI(d) mice, which do not undergo rebound thrombocytosis in response to 5FU, provide a genetic approach to the study of this phenomenon. Recent reports by several groups that the SI locus encodes a protein known variably as stem cell factor (SCF), mast cell growth factor, or kit ligand, suggests the possibility that the lack of wild-type SCF in SI/SI(d) mice is responsible for their defective response to 5FU-induced thrombocytopenia. It is shown in this report that SCF-treated SI/SI(d) mice are as capable as their wild-type littermates in undergoing rebound thrombocytosis. W/Wv mice, mutated at the locus encoding the SCF receptor, also do not undergo rebound thrombocytosis, but are not responsive to SCF treatment. In normal mice, it is shown by RNA solution hybridization that SCF mRNA expression is increased during the 5FU-induced platelet nadir period. It is also shown by autoradiography that maturing megakaryocytes express SCF receptors, and that in vivo administration of SCF significantly raises the numbers of megakaryocytes, as well as circulating platelet counts. Taken together, these data indicate that SCF may be an important regulator of platelet production under both normal and physiologically disturbed situations.

Purification and characterization of soluble forms of human and rat stem cell factor recombinantly expressed by Escherichia coli and by Chinese hamster ovary cells.

Stem cell factor (SCF) is a novel, early-acting hematopoietic factor. It was isolated from the medium of a rat cell line in a soluble, processed form (Zsebo et al., 1990, Cell 63, 195). The cloned human and rat genes encode the soluble form plus additional C-terminal amino acids including a hydrophobic transmembrane domain (Martin et al., 1990, Cell 63, 203). We have recombinantly expressed forms of human and rat SCF corresponding to the soluble, processed form in Escherichia coli and in Chinese hamster ovary (CHO) cells. After expression in E. coli, folding and oxidation of the SCF polypeptides are required. The SCFs expressed in CHO cells are secreted into the medium in active state and, like the natural SCF, are glycosylated. Purification of the recombinant SCFs is described. Biological and biochemical characterization includes activity toward responsive human and mouse cell lines, N-terminal amino acid sequences, disulfide bond linkages, and sites of glycosylation.

Stem cell factor increases colony-forming unit-spleen number in vitro in synergy with interleukin-6, and in vivo in Sl/Sld mice as a single factor.

Hematopoiesis is thought to be modulated by interactions of progenitor cells with hematopoietic growth factors. We have shown that colony-forming units-spleen (CFU-S) and repopulating stem cells require interleukin-3 (IL-3) to survive in vitro, and that CFU-S number and long-term repopulating ability can be increased by culture in the combination of IL-3 and IL-6. In this report, we describe the effects of stem cell factor (SCF) on CFU-S and repopulating stem cells. Injection of SCF into anemic Sl/Sld mice caused a twofold and 20-fold increase in CFU-S number in the bone marrow and spleen of treated animals, respectively. After 6 days in suspension culture, CFU-S number increased threefold in cultures supplemented with SCF and IL-6, or SCF, IL-3, and IL-6 relative to the number at day 0. The long-term repopulating ability of cells cultured in SCF, IL-3, and IL-6 was approximately sevenfold better than that of cells cultured in IL-3 or SCF. Similar experiments were performed on populations of bone marrow cells enriched for, or depleted of, CFU-S by elutriation and lineage subtraction. The combination of SCF and IL-6 increased CFU-S number approximately fourfold to eightfold in the CFU-S-enriched fraction, but had no effect on the CFU-S-depleted cells. These results show that SCF alone can increase CFU-S number in vivo, and in combination with other growth factors increases CFU-S numbers in vitro.

Isolation and characterization of a monoclonal antibody that recognizes the human c-kit receptor.

Stem cell factor (SCF) stimulates the growth of burst-forming unit-erythroid (BFU-E) and colony-forming unit granulocyte-macrophage (CFU-GM) by binding to a specific cell surface receptor. The receptor for SCF is encoded by the protooncogene c-kit. After immunizing mice with the human erythroleukemia cell line OCIM1, we obtained a monoclonal antibody (MoAb) that recognizes the human c-kit receptor. This MoAb, designated SR-1, blocks binding of 125I-human SCF to the c-kit receptor, and neutralizes the biologic effects of SCF in hematopoietic colony assays. With few exceptions, c-kit expression was identified on all hematopoietic and lymphoid cell lines tested by indirect immunofluorescent analysis using SR-1 and by binding studies with 125I-SCF. SR-1 recognizes a small fraction of normal bone marrow mononuclear cells, and these cells have the morphologic appearance of blasts. Colony assays show that BFU-E and CFU-GM display the c-kit receptor. SR-1 does not cross-react with murine c-kit protein, indicating that the binding epitopes of the human and murine c-kit receptors are antigenically distinct. This MoAb may be useful to characterize the spectrum of cells that display the c-kit receptor and to further define the role of SCF in hematopoiesis.

Identification, purification, and biological characterization of hematopoietic stem cell factor from buffalo rat liver--conditioned medium.

We have identified a novel growth factor, stem cell factor (SCF), for primitive hematopoietic progenitors based on its activity on bone marrow cells derived from mice treated with 5-fluorouracil. The protein was isolated from the medium conditioned by Buffalo rat liver cells. It is heavily glycosylated, with both N-linked and O-linked carbohydrate. Amino acid sequence following removal of N-terminal pyroglutamate is presented. The protein has potent synergistic activities in semisolid bone marrow cultures in conjunction with colony-stimulating factors. It is also a growth factor for mast cells. In two companion papers, we present the sequences of partial SCF cDNAs, identify SCF as a c-kit ligand, and map the SCF gene to the Sl locus of the mouse.

Stem cell factor is encoded at the Sl locus of the mouse and is the ligand for the c-kit tyrosine kinase receptor.

We have cloned a partial cDNA encoding murine stem cell factor (SCF) and show that the gene is syntenic with the Sl locus on mouse chromosome 10. Using retroviral vectors to immortalize fetal liver stromal cell lines from mice harboring lethal mutations at the Sl locus (Sl/Sl), we have shown that SCF genomic sequences are deleted in these lines. Furthermore, two other mutations at Sl, Sld and Sl12H, are associated with deletions or alterations of SCF genomic sequences. In vivo administration of SCF can reverse the macrocytic anemia and locally repair the mast cell deficiency of Sl/Sld mice. We have also provided biological and physical evidence that SCF is a ligand for the c-kit receptor.