Keith E. Langley

Stem cell factor is encoded at the SI 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.

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.

Primary structure and functional expression of rat and human stem cell factor DNAs.

Partial cDNA and genomic clones of rat stem cell factor (SCF) have been isolated. Using probes based on the rat sequence, partial and full-length cDNA and genomic clones of human SCF have been isolated. Based on the primary structure of the 164 amino acid protein purified from BRL-3A cells, truncated forms of the rat and human proteins have been expressed in E. coli and mammalian cells and have been shown to possess biological activity. SCF is able to augment the proliferation of both myeloid and lymphoid hematopoietic progenitors in bone marrow cultures. SCF exhibits potent synergistic activities in conjunction with colony-stimulating factors, resulting in increased colony numbers and colony size.

Tissue Inhibitor of Metalloproteinase-2 (TIMP-2) Binds to the Catalytic Domain of the Cell Surface Receptor, Membrane Type 1-Matrix Metalloproteinase 1 (MT1-MMP)

It has been proposed that tissue inhibitor of metalloproteinase-2 (TIMP-2), in stoichiometric concentrations, serves as an intermediate in progelatinase A activation by binding to activated membrane type 1-matrix metalloproteinase 1 (MT1-MMP) on the plasma membrane. An MT1-MMP-independent cell surface receptor for TIMP-2 has also been postulated. To clarify TIMP-2 binding, we have performed 125I-TIMP-2 binding studies on transfected COS-1 cells and endothelial cells. Specific receptors for TIMP-2 were identified on COS-1 cells transfected with MT1-MMP cDNA, but not on vector-transfected cells. Treatment of MT1-MMP transfected COS-1 cells with a hydroxamic acid inhibitor of MMPs, CT-1746, but not an inactive stereoisomer, CT-1915, produced dose-dependent inhibition of specific TIMP-2 binding comparable with that noted with excess unlabeled TIMP-2. This result suggests that TIMP-2 binds to the zinc catalytic site of MT1-MMP. As demonstrated by the limited competition for binding of C-terminal deleted TIMP-2, the C-terminal domain of TIMP-2 participates in binding to MT1-MMP. Cross-linking studies followed by immunoprecipitation using antibodies to MT1-MMP were employed to identify 125I-TIMP-2·MT1-MMP complexes in MT1-MMP-transfected COS-1 cell membrane extracts. TIMP-2 receptors were also identified on concanavalin A-treated human umbilical vein endothelial cells; inhibition of TIMP-2 binding with CT-1746 was demonstrated.

A cytokine-based neuroimmunologic mechanism of cancer-related symptoms.

While many of the multiple symptoms that cancer patients have are due to the disease, it is increasingly recognized that pain, fatigue, sleep disturbance, cognitive dysfunction and affective symptoms are treatment related, and may lead to treatment delays or premature treatment termination. This symptom burden, a subjective counterpart of tumor burden, causes significant distress. Progress in understanding the mechanisms that underlie these symptoms may lead to new therapies for symptom control. Recently, some of these symptoms have been related to the actions of certain cytokines that produce a constellation of symptoms and behavioral signs when given exogenously to both humans and animals. The cytokine-induced sickness behavior that occurs in animals after the administration of infectious or inflammatory agents or certain proinflammatory cytokines has much in common with the symptoms experienced by cancer patients. Accordingly, we propose that cancer-related symptom clusters share common cytokine-based neuroimmunologic mechanisms. In this review, we provide evidence from clinical and animal studies that correlate the altered cytokine profile with cancer-related symptoms. We also propose that the expression of coexisting symptoms is linked to the deregulated activity of nuclear factor-kappa B, the transcription factor responsible for the production of cytokines and mediators of the inflammatory responses due to cancer and/or cancer treatment. These concepts open exciting new avenues for translational research in the pathophysiology and treatment of cancer-related symptoms.

Structure of the active core of human stem cell factor and analysis of binding to its receptor kit.

Stem cell factor (SCF) is an early‐acting hematopoietic cytokine that elicits multiple biological effects. SCF is dimeric and occurs in soluble and membrane‐bound forms. It transduces signals by ligand‐ mediated dimerization of its receptor, Kit, which is a receptor tyrosine kinase related to the receptors for platelet‐derived growth factor (PDGF), macrophage colony‐stimulating factor, Flt‐3 ligand and vascular endothelial growth factor (VEGF). All of these have extracellular ligand‐binding portions composed of immunoglobulin‐like repeats. We have determined the crystal structure of selenomethionyl soluble human SCF at 2.2 Å resolution by multiwavelength anomalous diffraction phasing. SCF has the characteristic helical cytokine topology, but the structure is unique apart from core portions. The SCF dimer has a symmetric ‘head‐to‐head’ association. Using various prior observations, we have located potential Kit‐binding sites on the SCF dimer. A superimposition of this dimer onto VEGF in its complex with the receptor Flt‐1 places the binding sites on SCF in positions of topographical and electrostatic complementarity with the Kit counterparts of Flt‐1, and a similar model can be made for the complex of PDGF with its receptor.

The Majority of Stem Cell Factor Exists as Monomer under Physiological Conditions IMPLICATIONS FOR DIMERIZATION MEDIATING BIOLOGICAL ACTIVITY

Soluble Escherichia coli-derived recombinant human stem cell factor (rhSCF) forms a non-covalently associated dimer. We have determined a dimer association constant (Ka) of 2-4 × 108 M−1, using sedimentation equilibrium and size exclusion chromatography. SCF has been shown previously to be present at concentrations of approximately 3.3 ng/ml in human serum. Based on the dimerization Ka, greater than 90% of the circulating SCF would be in the monomeric form. When 125I-rhSCF was added to human serum and the serum analyzed by size exclusion chromatography, 72-49% of rhSCF was monomer when the total SCF concentration was in the range of 10-100 ng/ml, consistent with the Ka determination. Three SCF variants, SCF(F63C), SCF (V49L,F63L), and SCF(A165C), were recombinantly expressed in Escherichia coli, purified, and characterized. The dimer Ka values, biophysical properties, and biological activities of these variants were studied. Dimerization-defective variants SCF(F63C)S-CH2CONH2 and SCF(V49L,F63L) showed substantially reduced mitogenic activity, while the activity of the Cys165-Cys165 disulfide-linked SCF(A165C) dimer was 10-fold higher than that of wild type rhSCF. The results suggest a correlation between dimerization affinity and biological activity, consistent with a model in which SCF dimerization mediates dimerization of its receptor, Kit, and subsequent signal transduction.

Recombinant bovine somatotropin more than doubles the growth rate of coho salmon (Oncorhynchus kisutch) acclimated to seawater and ambient winter conditions

Abstract Sub-adult coho salmon ( Oncorhynchus kisutch ) were tested in a culture situation to assess whether somatotropin therapy would improve growth performance during their first “sea-winter”. Untreated fish and sham-treated fish did not differ significantly in their growth performance. Over the 12-week course of the experiment, the mean increase in fork length for these groups was 9.5% (±6.0 SD) while the mean increase in weight was 29.6% (±24.1 SD). Relative to these control groups, the administration of recombinant bovine somatotropin by injection, cholesterol pellet or mini-osmotic pump resulted in substantially better growth. Mean increases in fork length ranged from 16.5% (±5.5 SD) to 23.0% (±8.7 SD) while mean increases in weight ranged from 52.2% (±31.2 SD) to 65.5% (±44.0 SD). Specific growth rate was elevated 2–4 weeks after the initiation of somatotropin therapy and the effect did not diminish over the 8-week treatment period. Subsequent to hormone withdrawal, the growth rates of somatotropin-treated fish were no longer distinguishble from those of the controls. Somatotropin was noted to decrease the rate of growth depensation, yielding a more uniform group of salmon with respect to size. Condition factor was decreased by the hormone treatment. It was concluded that somatotropin treatment can produce a significant increase in the growth rate of coho salmon, both after they have made the transition to salt water and during the winter months, when environmental conditions are sub-optimal for growth.

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.

Matrix metalloproteinases and their inhibitors in tumor progression.

A characteristic feature of malignant cells is their ability to invade the surrounding tissues and to form new metastatic tumors in distant organs. Metastasis is the major life-threatening aspect of cancer and the major cause of failure to maintain longterm disease-free remission in cancer patients. When a cancer has disseminated, it is almost always resistant to conventional treatment. Over the last two decades significant efforts have been made to understand the molecular and cellular mechanisms involved in tumor dissemination, with the anticipation that this information may lead to the identification of innovative approaches to control this process. Studies have shown that the metastatic process is under genetic control separate from tumorigenesis and that alteration of several genes is required. Emphasis has been placed on the interaction between tumor cells and the extracellular matrix (ECM), and on the many changes that perturb the harmony between cells and their extracellular environment during tumor invasion (reviewed in ref. 1). Degradation of matrix proteins is an important step that allows tumor cells to penetrate the ECM, to intravasate into blood vessels, and to extravasate at distant sites. In addition, because the ECM is also a reservoir of many factors, including growth factors, it is anticipated that proteolytic degradation of the matrix will significantly affect the equilibrium between cells, matrix proteins to which they are attached, and matrix bound growth factors for which they have specific membrane receptors. Since 1972, when collagcnase activity in cancer was first described,zJ a growing number of reports have pointed to the key role of matrix metalloproteinases (MMPs) in this ~ g a r d . ~ . ~ These proteases, which now comprise a large family, are secreted as inactive proenzymes which can subsequently become activated (reviewed in ref. 6). Several lines of evidence have implicated the MMPs in cancer cell tumorigenicity, invasion, and metastasis, as outlined in TABLE 1.This manuscript reviews these lines of evidence,