Role for the stem cell factor/KIT complex in Schwann cell neoplasia and mast cell proliferation associated with neurofibromatosis.

Schwann cells are the primary cell type in the disfiguring lesions associated with neurofibromatosis type 1 (NF-1). These lesions also contain abnormally high numbers of mast cells, a cell type which develops in response to stem cell factor. We report here that neonatal and adult rat and human Schwann cells, as well as a transfected rat Schwann cell line and a human Schwannoma line derived from an NF-1 patient, all produced stem cell factor mRNA and protein. In coculture experiments, surface expression of stem cell factor by neonatal rat Schwann cells was profoundly downregulated by contact with dorsal root ganglion neurites. The receptor for stem cell factor, KIT, was not expressed in normal Schwann cells but was expressed in the human Schwannoma line, suggesting that aberrant KIT expression may form an autocrine loop in certain Schwann cell neoplasias.

Expression of c-kit by mesenteric lymph node cells from Nippostrongylus brasiliensis-infected mice and by mast cell colonies developing from these cells in response to 3T3 fibroblast-conditioned medium.

Mast cell committed progenitors are nongranulated cells found in mesenteric lymph nodes of mice infected with Nippostrongylus brasiliensis (Nb-MLN) but not from normal mice. Mast cell committed progenitors can respond to either IL-3 or to a factor(s) present in 3T3 fibroblast conditioned media (F-CM) by formation of mast cell colonies. Previous studies from ours and other laboratories suggested that mast cell differentiation involved the W allele product, c-kit, as a receptor and Sl allele product, stem cell factor, as a growth factor. We report here that Nb-MLN cells, which can respond to F-CM by mast cell colony formation, also contain cells that express message for c-kit, and that c-kit message cannot be detected in naive mesenteric lymph node cells, which cannot respond to F-CM. Antisense oligonucleotides to c-kit inhibit mast cell colony formation by Nb-MLN cells in response to F-CM, but not to conditioned medium of PWM-stimulated spleen cells as a source of IL-3. The antisense oligonucleotides also inhibit the degree of granulation by mast cells derived from culture. The results suggest that c-kit and its ligand, stem cell factor, are necessary for mast cell-committed progenitors to proliferate and granulate in response to F-CM but not IL-3.

c-kit expression by CD34+ bone marrow progenitors and inhibition of response to recombinant human interleukin-3 following exposure to c-kit antisense oligonucleotides.

The c-kit proto-oncogene encodes a receptor having tyrosine-specific kinase activity and has been mapped to chromosome 4 in the human and chromosome 5 in the mouse, at the dominant white spotting locus (W). Mutations at the W locus affect various aspects of murine hematopoiesis. The c-kit proto-oncogene has been shown to be expressed by leukemic myeloblasts, but not by normal unseparated human bone marrow cells. The role of this oncogene in differentiation and proliferation of human hematopoietic progenitors is presently undefined. To determine c-kit expression by normal hematopoietic progenitors, CD34+ cells were isolated from disease-free human bone marrow, and RNA-based polymerase chain reaction (PCR) techniques were used to assess expression. By this method, we have demonstrated c-kit expression by CD34+ bone marrow progenitors. To address the functional requirement for c-kit expression in normal human hematopoiesis, CD34+ cells were incubated in the presence of sense, antisense, or missense oligonucleotides to c-kit, and subsequently cultured in the presence of either recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) or recombinant human interleukin-3 (rhIL-3). Exposure of CD34+ cells to c-kit antisense oligonucleotides significantly inhibited colony-forming ability of cells cultured in the presence of rhIL-3, but had no effect on colony formation of cells cultured in rhGM-CSF. Together, these data suggest a possible role for c-kit in hematopoietic proliferation and differentiation that may be linked to some, but not all, stimulatory factors.

Initial characterization of a cytokine which induces differentiation and cytolytic activity in HL-60 promyelocytic leukemia cells: evidence that the cytokine is distinct from other known differentiation-active cytokines.

In this paper we report that, like dimethyl sulfoxide (DMSO), retinoic acid (RA), and conditioned medium (CM) from lectin-stimulated mononuclear leukocytes, CM from a human null cell leukemia line (Reh) induces HL-60 promyelocytic leukemia cells to respond in an enhanced manner to phorbol diester (PDE). Furthermore, Reh-CM induces PDE-resistant HL-60-1E3 cells to respond to PDE and lyse target cells. Additionally, both HL-60 and HL-60-1E3 cells exposed to Reh-CM for 3 days produce superoxide anion and express cell surface antigens present on mature mononuclear phagocytes. No colony-stimulating factor (CSF) or interferon (IFN) activity was detected in Reh-CM, and differentiation activity (DA) was not removed from Reh-CM by insolubilized anti-IFN gamma. While Reh-CM is antiproliferative against a panel of cell lines, its spectrum of activity is different than tumor necrosis factor (TNF) alpha, and neither TNF alpha nor TNF beta inhibit proliferation of HL-60-1E3 or induce these cells to respond to PDE. The differentiation factor (DF) material has been partially purified by ammonium sulfate precipitation and is non-dialyzable; unstable to heat, acid, or alkali treatment; and the activity is not blocked by anti-IL-6 or anti-IFN alpha. The data presented in this paper suggest the presence of a differentiation-inducing factor which is distinct from CSF, IFN alpha or -gamma, TNF alpha, or -beta, or IL-6, which may play a role in the differentiation of malignant (leukemic) and normal cells of the myelomonocytic lineage.

P48 induces tumor necrosis factor and IL-1 secretion by human monocytes.

Bacterial products are potent stimulators of TNF and IL-1 release, however, the factors that regulate cytokine secretion in the absence of bacterial products are not well defined. P48 is a cytokine recently identified in the supernatant of the human null cell leukemia cell line Reh, which induces differentiation and cytolytic activity in HL-60 cells. P48 has been purified to homogeneity and is distinct from TNF-alpha TNF-beta, IFN-gamma, IL-6, and macrophage CSF. In the present study we examined the ability of P48 to stimulate cytokine release by human peripheral blood monocytes. P48 stimulated the secretion of TNF and IL-1 in a dose-dependent manner. Priming the monocytes with IFN-gamma enhanced P48-induced cytokine release but was not a requirement for secretion. Cytokine secretion was in response to P48 and was not caused by endotoxin contamination. The cytokine-inducing activity of P48 was extremely sensitive to heat treatment but could not be eliminated by using polymyxin B. Polyclonal antisera to P48 completely blocked the cytokine-inducing activity. P48 may be an important new member of the cytokine network involved in the regulation of cytokine secretion by monocytes.

Purification and further characterization of a non-tumor necrosis factor alpha or beta differentiation-inducing cytokine, P48.

In this report, we present the further characterization and purification of a cytokine differentiation factor, termed P48, which unlike previously described differentiation factors is antigenically unrelated to tumor necrosis factor alpha (TNF-alpha), tumor necrosis factor beta (TNF-beta), and gamma interferon. HL-60 cells and phorbol diester-resistant HL-60-1E3 cells exposed to conditioned medium from Reh cells mature along the monocyte/macrophage pathway, as assessed by several assays (express nonspecific esterase, produce superoxide anion, morphologically resemble monocytes, mediate phorbol diester-triggered extracellular cytolytic activity). Reh cell conditioned medium is antiproliferative toward a panel of cell lines, is not nonspecifically cytotoxic, has no antiviral or colony-stimulating factor activities, and is not affected by exposure to insolubilized anti-gamma interferon. A 48-kDa glycoprotein (P48) which mediates this differentiation factor activity has been purified to homogeneity from Reh cell conditioned medium, and a polyclonal neutralizing antiserum has been produced. P48 activity is not blocked by either anti-TNF-alpha and anti-TNF-beta and on Western blot analysis is antigenically distinct from TNF-alpha and TNF-beta. In addition, polyclonal anti-P48 does not block either TNF-alpha or TNF-beta activities or recognize either on Western blots. Unlike gamma interferon, colony-stimulating factor, TNF-alpha, or TNF-beta, P48 reverses phorbol diester resistance of HL-60-1E3 cells. These studies present strong evidence for the existence of a previously unrecognized cytokine which, unlike other reported differentiation factors, is antigenically unrelated to TNF-alpha or TNF-beta. P48 may play an important role in growth and development of normal and abnormal (leukemic) hematopoietic and nonhematopoietic cells.

Altered regulation of c-myc in an HL-60 differentiation resistant subclone, HL-60-1E3.

HL-60 cells treated with phorbol dibutyrate (PDBu) differentiate into cells which functionally and morphologically resemble macrophages (G. Rovera, D. Santoli, and D. Damskey, Proc. Natl. Acad. Sci. USA, 75: 2779-2783, 1979; E. Huberman and M.F. Callahan, Proc. Natl. Acad. Sci. USA, 76:1293-1298, 1979). This differentiation involves modulation of the expression of several cellular oncogenes. However, the significance of the temporal relationships between differentiation events and specific oncogene expression are not known. Others have reported that transcriptional down regulation of c-myc occurs early in the differentiation of HL-60 cells (R.D. Dalla-Favera et al., Haematol. Blood Transfusion, 28: 247-253, 1983; L.E. Grosso and H.C. Pitot, Biochem. Biophys. Res. Commun., 119: 473-480, 1984). To determine the significance of the regulation of c-myc during HL-60 maturation, we performed parallel PDBu induction studies analyzing the kinetics of expression of c-myc, cell cycle frequency distribution, cytotoxic effector activity, and clonogenic potential in HL-60 cells and in a partial-differentiation resistant HL-60 clone (HL-60-1E3) (J. A. Leftwich, P. Carlson, B. Adelman, and R. E. Hall, Cancer Res., 47: 1319-1324, 1987). We report that PDBu stimulation results in early c-myc transcriptional down regulation in the HL-60-1E3 clone cells with the same kinetics as has been previously reported for HL-60 parental cells (R. D. Dalla-Favera et al., Haematol. Blood Transfusion, 28: 247-253, 1983; L. E. Grosso and H. C. Pitot, Biochem. Biophys. Res. Commun., 119: 473-480, 1984). However, reexpression of c-myc occurs 15 hours postinduction in HL-60-1E3 but not parental cells. This reexpression is maintained through 30 h of stimulation and correlates with a lack of terminal commitment as assessed by an increase in clonogenic potential and the inability of these cells to acquire cytotoxic function. Sequential stimulation of HL-60-1E3 cells with DMSO and PDBu overcomes the block in macrophage differentiation (J. A. Leftwich, P. Carlson, B. Adelman, and R. E. Hall, Cancer Res., 47; 1319-1324, 1987). Such treatment results in a transient reexpression of c-myc at 15 h after PDBu treatment, and the complete downregulation of c-myc 24 h postinduction. These data suggest that the reported early decrease in c-myc transcripts following PDBu stimulus in HL-60 cells is not sufficient to commit these cells to macrophage-like terminal differentiation. Late regulation of c-myc gene expression may be an important additional component of the regulatory mechanisms which allow HL-60 cells to complete this program.

HL-60-1E3, a novel phorbol diester-resistant HL-60 cell line.

The HL-60 (human promyelocytic leukemia) cell line has proved to be a useful model for the study of the expression of cellular functions and markers associated with hematopoietic differentiation. We report here the development and initial characterization of a novel, differentiation-resistant HL-60 cell line (HL-60-1E3) which was established by cloning the parent HL-60 line in the absence of mutagens or differentiation-inducing agents. HL-60-1E3 exhibits markedly reduced phorbol diester-induced expression of extracellular cytolytic activity, nonspecific esterase, phagocytosis, and surface Mo1 antigen. In addition, dimethyl sulfoxide-induced expression of both Mo1 and Mo2 is markedly reduced. However, if HL-60-1E3 is exposed to dimethyl sulfoxide, it acquires appreciable phorbol diester-triggered cytolytic activity and production of superoxide anion (O2-). Phorbol diester receptor number and dissociation constant (Kd) obtained by Scatchard analysis are not significantly different for the two cell lines. The HL-60-1E3 cell line should provide a useful adjunct to other cell lines used in the study of normal myeloid and leukemic cell differentiation, as well as the study of cytokine maturation factors and oncogene expression.

Enhancement of phorbol diester-induced HL-60-mediated cytotoxicity by retinoic acid, dimethyl sulfoxide, and 5-azacytidine.

Both peripheral blood monocytes and neutrophils are known to be capable of lysing a variety of extracellular tumor and non-tumor cell targets. The HL-60 human promyelocytic leukemia cell line has served as a useful model of human granulocyte and macrophage differentiation in studies from many laboratories. We have previously reported that phorbol diesters, which induce differentiation along the macrophage pathway, stimulate HL-60 cells to become strikingly cytotoxic to a variety of red cell targets. We now report that agents known to differentiate HL-60 along the granulocyte pathway (retinoic acid, dimethyl sulfoxide, 5-azacytidine) do not, in themselves, induce HL-60 to become cytotoxic. However, previous exposure (3-5 days) to these granulocyte pathway active agents markedly enhances phorbol diester-triggered killing. This enhancement is particularly striking at decreased effector:target ratios (as low as one effector per five targets) and is also demonstrated by a shift to lower concentrations of the phorbol diester dose-response curve. Retinoic acid is the most effective of the three agents tested, although priming (previous exposure) with dimethyl sulfoxide or 5-azacytidine also markedly enhances killing. These studies demonstrate that HL-60-mediated killing may be dissected pharmacologically into at least two distinct steps and further support the utility of this model system in studies of the development of macrophage-like cytotoxic cells. This system has also proven to be useful in the characterization of cytokines which mimic the differentiation effects of retinoic acid and dimethyl sulfoxide (J. A. Leftwich and R. E. Hall, manuscript in preparation).