The product of the c-fms proto-oncogene: a glycoprotein with associated tyrosine kinase activity.

The c-fms proto-oncogene is a member of a gene family that has been implicated in tumorigenesis. Glycoproteins encoded by c-fms were identified in cat spleen cells by means of an immune-complex kinase assay performed with monoclonal antibodies to v-fms-coded epitopes. The major form of the normal cellular glycoprotein has an apparent molecular weight of 170,000 and, like the product of the viral oncogene, serves as a substrate for an associated tyrosine-specific protein kinase activity in vitro. The results suggest that the transforming glycoprotein specified by v-fms is a truncated form of a c-fms-coded growth factor receptor.

Transfer and expression of the gene encoding a human myeloid membrane antigen (gp150).

DNA from the human myeloid cell line HL-60 was cotransfected with the cloned thymidine kinase (tk) gene of herpes simplex virus into tk-deficient mouse L cells. tk-positive recipients expressing antigens detected on HL-60 cells were isolated with a fluorescence-activated cell sorter by use of a panel of monoclonal antibodies that detect epitopes on both normal and malignant myeloid cells. Independently sorted populations of transformed mouse cells showed concordant reactivities with four of the monoclonal antibodies in the panel (DU-HL60-4, MY7, MCS.2, and SJ-D1), which suggested that these antibodies reacted to products of a single human gene. A second round of DNA transfection and cell sorting was performed with donor DNA from primary transformants. Two different dominant selection systems were used to isolate secondary mouse L cell and NIH/3T3 cell transformants that coexpressed the same epitopes. Analysis of cellular DNA from secondary mouse cell subclones with a probe specific for human repetitive DNA sequences revealed a minimal human DNA complement containing a characteristic set of restriction fragments common to independently derived subclones. Two glycoproteins, of 130,000 (gp130) and 150,000 (gp150) mol wt, were specifically immunoprecipitated from metabolically labeled lysates of mouse cell transformants and were shown to contain [35S]methionine-labeled tryptic peptides identical to those of analogous glycoproteins expressed in the donor human myeloid cell line. Kinetic and biochemical analyses established that gp130 is a precursor that differs in its carbohydrate moiety from gp150, the mature form of the glycoprotein detected on the cell surface. The isolation of human gene sequences encoding gp150 in a mouse cell genetic background provides the possibility of molecularly cloning the gene and represents a general strategy for isolating human genes encoding differentiation-specific cell surface antigens.

Expression of the human c-fms proto-oncogene product (colony-stimulating factor-1 receptor) on peripheral blood mononuclear cells and choriocarcinoma cell lines.

The c-fms gene product is related, and possibly identical, to the receptor for the mononuclear phagocyte colony stimulating factor, CSF-1. Using antisera to a recombinant v-fms--coded polypeptide, glycoproteins encoded by the human c-fms locus were detected in mononuclear cells from normal peripheral blood and in promyelocytic HL-60 cells 24 h after induction of monocytic differentiation with phorbol ester. The 150-kD human c-fms--coded glycoprotein was expressed at the cell surface, was active as a tyrosine-specific protein kinase in vitro, and shared primary structural features with the product of the feline retroviral v-fms oncogene. A biochemically indistinguishable glycoprotein was detected in human choriocarcinoma cell lines. Like peripheral blood mononuclear cells and phorbol ester-treated HL-60 cells, the choriocarcinoma cells expressed high affinity binding sites for human CSF-1. In addition to serving as a lineage specific growth factor in hematopoiesis, CSF-1 may play a role in normal trophoblast development.

Differential processing of colony-stimulating factor 1 precursors encoded by two human cDNAs.

The biosynthesis of macrophage colony-stimulating factor 1 (CSF-1) was examined in mouse NIH-3T3 fibroblasts transfected with a retroviral vector expressing the 554-amino-acid product of a human 4-kilobase (kb) CSF-1 cDNA. Similar to results previously obtained with a 1.6-kb human cDNA that codes for a 256-amino-acid CSF-1 precursor, the results of the present study showed that NIH-3T3 cells expressing the product of the 4-kb clone produced biologically active human CSF-1 and were transformed by an autocrine mechanism when cotransfected with a vector containing a human c-fms (CSF-1 receptor) cDNA. The 4-kb CSF-1 cDNA product was synthesized as an integral transmembrane glycoprotein that was assembled into disulfide-linked dimers and rapidly underwent proteolytic cleavage to generate a soluble growth factor. Although the smaller CSF-1 precursor specified by the 1.6-kb human cDNA was stably expressed as a membrane-bound glycoprotein at the cell surface and was slowly cleaved to release the extracellular growth factor, the cell-associated product of the 4-kb clone was efficiently processed to the secreted form and was not detected on the plasma membrane. Digestion with glycosidic enzymes indicated that soluble CSF-1 encoded by the 4-kb cDNA contained both asparagine(N)-linked and O-linked carbohydrate chains, whereas the product of the 1.6-kb clone had only N-linked oligosaccharides. Removal of the carbohydrate indicated that the polypeptide chain of the secreted 4-kb cDNA product was longer than that of the corresponding form encoded by the smaller clone. These differences in posttranslational processing may reflect diverse physiological roles for the products of the two CSF-1 precursors in vivo.

Ligand-induced tyrosine kinase activity of the colony-stimulating factor 1 receptor in a murine macrophage cell line.

Metabolic labeling of simian virus 40-immortalized murine macrophages with 32Pi and immunoblotting with antibodies to phosphotyrosine demonstrated that the c-fms proto-oncogene product (colony-stimulating factor 1 [CSF-1] receptor) was phosphorylated on tyrosine in vivo and rapidly degraded in response to CSF-1. Stimulation of the CSF-1 receptor also induced immediate phosphorylation of several other cellular proteins on tyrosine. By contrast, the mature cell surface glycoprotein encoded by the v-fms oncogene was phosphorylated on tyrosine in the absence of CSF-1, suggesting that it functions as a ligand-independent kinase.

Cell surface expression of v-fms-coded glycoproteins is required for transformation.

The viral oncogene v-fms encodes a transforming glycoprotein with in vitro tyrosine-specific protein kinase activity. Although most v-fms-coded molecules remain internally sequestered in transformed cells, a minor population of molecules is transported to the cell surface. An engineered deletion mutant lacking 348 base pairs of the 3.0-kilobase-pair v-fms gene encoded a polypeptide that was 15 kilodaltons smaller than the wild-type v-fms gene product. The in-frame deletion of 116 amino acids was adjacent to the transmembrane anchor peptide located near the middle of the predicted protein sequence and 432 amino acids from the carboxyl terminus. The mutant polypeptide acquired N-linked oligosaccharide chains, was proteolytically processed in a manner similar to the wild-type glycoprotein, and exhibited an associated tyrosine-specific protein kinase activity in vitro. However, the N-linked oligosaccharides of the mutant glycoprotein were not processed to complex carbohydrate chains, and the glycoprotein was not detected at the cell surface. Cells expressing high levels of the mutant glycoprotein did not undergo morphological transformation and did not form colonies in semisolid medium. The transforming activity of the v-fms gene product therefore appears to be mediated through target molecules on the plasma membrane.

Synthesis of membrane-bound colony-stimulating factor 1 (CSF-1) and downmodulation of CSF-1 receptors in NIH 3T3 cells transformed by cotransfection of the human CSF-1 and c-fms (CSF-1 receptor) genes.

NIH 3T3 cells cotransfected with the human c-fms proto-oncogene together with a 1.6-kilobase cDNA clone encoding a 256-amino-acid precursor of the human mononuclear phagocyte colony-stimulating factor CSF-1 (M-CSF) undergo transformation by an autocrine mechanism. The number of CSF-1 receptors on the surface of transformed cells was regulated by ligand-induced receptor degradation and was inversely proportional to the quantity of CSF-1 produced. A tyrosine-to-phenylalanine mutation at position 969 near the receptor carboxyl terminus potentiated its transforming efficiency in cells cotransfected by the CSF-1 gene but did not affect receptor downmodulation. CSF-1 was synthesized as an integral transmembrane glycoprotein that was rapidly dimerized through disulfide bonds. The homodimer was externalized at the cell surface, where it underwent proteolysis to yield the soluble growth factor. Trypsin treatment of viable cells cleaved the plasma membrane form of CSF-1 to molecules of a size indistinguishable from that of the extracellular growth factor, suggesting that trypsinlike proteases regulate the rate of CSF-1 release from transformed cells. The data raise the possibility that this form of membrane-bound CSF-1 might stimulate receptors on adjacent cells through direct cell-cell interactions.

Proteolytic processing of a plasma membrane-bound precursor to human macrophage colony-stimulating factor (CSF-1) is accelerated by phorbol ester.

A transmembrane precursor to human macrophage colony-stimulating factor (M-CSF, CSF-1) is stably expressed at the cell surface where it is slowly and inefficiently cleaved to yield a soluble form of the growth factor. Incubation in the presence of phorbol ester resulted in rapid cleavage of the plasma membrane-bound precursor and release of soluble CSF-1. Within 60 min after phorbol treatment the quantity of growth factor recovered in the medium was more than 30-fold greater than that observed in the absence of the agent. The growth factor released in the presence of phorbol was biologically active and exhibited the same electrophoretic mobility as that obtained in the absence of the drug. Phorbol ester-accelerated processing of the cell surface CSF-1 precursor was abrogated by long-term exposure to phorbol, but was not inhibited by pretreatment with cycloheximide or incubation in serum-free medium. These results suggest that the enhanced post-translational processing of the CSF-1 precursor resulted from activation of a pre-existing cellular protease via a mechanism involving phorbol ester-mediated stimulation of protein kinase C.

Osteogenic sarcoma metastatic to the heart.

Of 20 patients with fatal osteogenic sarcoma who underwent postmortem examination, four had metastases to the heart. One had recurrent ventricular tachycardia, and one had anatomic evidence of aortic regurgitation due to massive periaortic neoplastic infiltration that prevented coaptation of the aortic cusps during ventricular diastole, one had massive invasion of the peri-superior vena caval syndrome, and one had neoplastic obstruction of the inferior vena cava as it entered the thorax. Among patients with either primary or secondary neoplasms to the heart, osteogenic sarcoma is unique because the metastases contain bone and, therefore, may be radiographically visible; they are usually large and often intracavitary.

The mononuclear phagocyte colony-stimulating factor (CSF-1, M-CSF).

The macrophage colony-stimulating factor-1 (CSF-1, M-CSF) induces the proliferation and supports the survival of mononuclear phagocytes and mediates its pleiotropic actions by binding to cell surface receptors encoded by the c-fms proto-oncogene. The structure, biologic activities, and therapeutic potential of CSF-1 and the role of its receptor in signal transduction in normal and transformed cells are discussed.

The fms gene and the CSF-1 receptor.

The c-fms proto-oncogene encodes an integral transmembrane glycoprotein with tyrosine specific protein kinase activity whose properties resemble those of receptors for polypeptide growth factors. The relatively restricted expression of the feline c-fms gene in mononuclear phagocytes (peripheral blood monocytes and tissue macrophages) and their committed bone marrow progenitors suggested that c-fms encoded a receptor for a macrophage specific growth factor. We found that the receptor for the mononuclear phagocyte colony stimulating factor, CSF-1 (M-CSF), is biochemically and immunologically related to the c-fms gene product, consistent with the hypothesis that c-fms is identical to the CSF-1 receptor gene (Sherr et al, 1985). Although the activity of CSF-1 has been defined through its action on haemopoietic cells, the c-fms gene is expressed in human placenta (Müller et al, 1983a) and in human choriocarcinoma cell lines derived from placental trophoblasts (Müller et al, 1983b). The latter cell lines exhibit binding sites for CSF-1, suggesting that this colony stimulating factor might also have an embryological role in placental development. The retroviral oncogene v-fms is a potent fibroblast transforming gene, whereas c-fms can be expressed at relatively high levels in certain normal tissues without causing neoplastic transformation. The glycoprotein encoded by v-fms is closely related to the c-fms gene product but differs at its extreme carboxy terminal end. Because v-fms, like c-fms, encodes a competent ligand binding domain, cells producing the v-fms gene product acquire the ability to bind CSF-1. Moreover, many fibroblast cell lines susceptible to transformation by v-fms produce the growth factor. Although this raises the possibility that v-fms transforms cells by an autocrine mechanism, antibodies to epitopes in the v-fms-coded ligand binding domain that interfere with CSF-1 binding, or antibodies to CSF-1 itself, do not affect the transformed phenotype. In membrane preparations, tyrosine-specific phosphorylation of the v-fms product appears to be constitutive, whereas in vitro phosphorylation of the c-fms-coded glycoprotein on tyrosine is enhanced in the presence of CSF-1. These results are most compatible with the possibility that critical alterations in the 3' coding region of the c-fms gene activate its kinase activity and unmask its latent transforming potential. An implication of these findings is that chromosomal rearrangements affecting c-fms could contribute to myeloid leukaemogenesis.

Abortive infection of lysogenic Bacillus subtilis 168(SPO2) by bacteriophage phi 1.

Virulent bacteriophage phi1 was not able to productively infect strains of Bacillus subtilis which were lysogenic for the temperate bacteriophage SPO2, although it adsorbed to, penetrated, and killed these bacteria. Studies of phage and host nucleic acid production in the nonpermissive host demonstrated that normal phi1 transcription was initiated early in the latent period, but this was followed by a general failure of host and phage nucleic acid synthesis about 10 to 15 min after infection. Mixed infections of phi1 and SPO2c(1), a clear-plaque mutant of SPO2, indicated that a similar inhibition of phi1 development occurred when this phage infected nonlysogenic B. subtilis cells committed to the SPO2c(1) lytic cycle. It is proposed that the SPO2- and SPO2c(1)-mediated interference did not act directly on the phi1 genome, but rather these phages altered the host physiology in such a manner that some normal step in phi1 development triggered a collapse of vital metabolic activities.

Prophage-mediated interference affecting the development of Bacillus subtilis bacteriophage phi e.

Bacteriophage phie shows a reduced efficiency of plating on strains of Bacillus subtilis which are lysogenic for the temperate bacteriophage SP02. Although this phenomenon resembles prophage-mediated restriction observed in other bacteria, host-controlled modification of phie was not observed. Mutants of phie which plated with high efficiency on the lysogenic host were isolated.

Structural protein markers in the avian oncoviruses.

The proteins of purified avian oncoviruses were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and isoelectric focusing. Certain members of the avian leukosis-sarcoma viruses (ALSV) had group-specific antigens with altered electrophoretic properties. (i) The p27 protein of Rous-associated virus 0 (RAV-0) had a lower electrophoretic mobility in SDS gels and a lower isoelectric point than the p27 of other ALSV. (ii) The p19 proteins of RAV-1, RAV-2, and the Bryan high-titer strain of Rous sarcoma virus had higher mobilities in SDS gels than did the corresponding protein of other viruses. This altered electrophoretic mobility was correlated with specific differences in the tryptic peptides of radioiodinated p19s. (iii) The p15 protein of RAV-7 had a lower mobility in SDS gels than did the p15 of other ALSV. These markers were used in a study of the structural proteins of subgroup E RAV-60 produced after infection of chicken embryo cells by exogenous ALSV. Although exogenous group-specific protein markers could often be identified in the subgroup E isolates, one RAV-60 had a p27 that comigrated with the p27 of RAV-0. The p19s of two other RAV-60 isolates had electrophoretic properties that were different than those of p19s from either RAV-0 or the exogenous viruses. These results support the hypothesis that RAV-60 is generated by recombination between endogenous and exogenous oncoviruses and indicate that at least the p27 encoded by RAV-0 is closely related to a protein specified by endogenous viral information in chicken cells.

Viral mutation affecting bacteriophage phi 1 development in Bacillus subtilis 168.

Bacillus subtilis bacteriophage phi 1m, a host-range variant, was isolated after mutagenesis of virulent bacteriophage phi 1. Unlike its wild-type antecedent, phi 1m could not form plaques on lawns of B subtilis 168 at 37 C, although it adsorbed to, penetrated, and killed this bacterium. Experiments conducted in liquid medium at 37 C showed that B. subtilis 168 cells allowed reduced levels of phi 1m development at low multiplicities of infection, whereas high multiplicity infections of this strain by the phage were abortive. Certain mutants, derived originally from B. subtilis 168, were observed to be permissive for phi 1m at 37 C; moreover, their permissive phenotype could be duplicated by growing wild-type B. subtilis 168 cells at temperatures above 47 C. Studies on phi 1m and host nucleic acid synthesis under nonpermissive conditions demonstrated that transciption and DNA synthesis proceeded up to 20 min after infection, after which time there was a cessation of all nucleic acid production. These observations are discussed with respect to other abortive bacteriophage infections in B. subtilis.

Formation of Rous associated virus-60: origin of the polymerase gene.

The DNA of normal chicken embryos contains sequences related to the avian leukosis-sarcoma viruses. RNA-dependent DNA polymerase of these viruses is encoded by a genetic element known as the pol gene. The nature of the endogenous virus pol gene in chicken cells was investigated by testing its ability to participate in genetic recombination. Rous-associated virus-60-type recombinant viruses isolated after infection of chicken cells with strains tsLA337PR-B or tsNY21SR-A, both of which produce a temperature-sensitive DNA polymerase, also possessed the temperature-sensitive lesion. These results are consistent with the hypothesis that the endogenous viral information used for the generation of Rous-associated virus-60 is deficient in at least part of the pol gene and that the defect includes that portion represented by the lesions in NY21 and LA337. The frequency of polymerase-negative BH-Rous sarcoma virus alpha formation was not affected by the levels of endogenous viral expression, which suggests that the alpha defect is not derived from the endogenous pol gene.

The role of temperate bacteriophage SP beta in prophage-mediated interference in Bacillus subtilis.

Virulent bacteriophage phi 1 grows on a variety of Bacillus subtilis strains, mutants of this virus which abortively infect the transformable bacillus. B. subtilis 168, while retaining the ability to productively infect related bacteria have been found. In the present study, we demonstrate that the inability of one such variant, phi 1m, to develop normally in strain 168 is mediated by cryptic prophage SP beta. The latter is a temperate bacteriophage which is carried by B. subtilis 168 and most strains derived from this bacterium. Phi 1 m infection of SP beta lysogens begins with apparently normal adsorption, penetration, and inititaion of virus-directed syntheses. At about the 20th min of the latent period, however, there is an abrupt cessation of nucleic acid synthesis and cellular respiration, accompanied by a change in cell permeability. This course of events can be altered to a permissive infection by mutation in the mpi gene of SP beta, by mutation in the spoOA gene of the host, or by growing SP beta lysogens at high temperature. In addition, we found a second class of phi 1 mutants which abortively infect B. subtilis 168 derivatives even in the absence of the SP beta prophage.

Direct stimulation of cells expressing receptors for macrophage colony-stimulating factor (CSF-1) by a plasma membrane-bound precursor of human CSF-1.

Secreted forms of macrophage colony-stimulating factor (M-CSF or CSF-1) are generated by proteolytic cleavage of membrane-bound glycoprotein precursors. Alternatively spliced transcripts of the human CSF-1 gene encode at least two different transmembrane precursors that are differentially processed in mammalian expression systems. The larger precursor rapidly undergoes proteolysis to yield the secreted growth factor and does not give rise to forms of CSF-1 detected on the cell surface. By contrast, the smaller human CSF-1 precursor is stably expressed on the plasma membrane where it is inefficiently cleaved to release a soluble molecule. To determine whether the smaller precursor is biologically active on the cell surface, mouse NIH-3T3 fibroblasts expressing the different forms of human CSF-1 were killed by chemical fixation and tested for their ability to support the proliferation of cells that require this growth factor. Only fixed cells expressing human CSF-1 precursors on their surface stimulated the growth in vitro of a murine macrophage cell line or normal mouse bone marrow-derived mononuclear phagocytes. The ability of these nonviable fibroblasts to induce the proliferation of CSF-1-dependent cells was not mediated by release of soluble growth factor, required direct contact with the target cells, and was blocked by neutralizing antiserum to CSF-1. These results demonstrate that the cell surface form of the human CSF-1 precursor is biologically active and indicate that plasma membrane-bound growth factors can functionally interact with receptor-bearing targets by direct cell-cell contact.

Prostaglandin E2 inhibition of growth in a colony-stimulating factor 1-dependent macrophage cell line.

The effects of prostaglandin E2 (PGE2) were examined in a murine macrophage cell line (BAC1.2F5) that was completely dependent on colony-stimulating factor-1 (CSF-1) for both growth and survival. The addition of PGE2 to cultures of BAC1.2F5 cells resulted in the inhibition of CSF-1-induced [3H]thymidine incorporation and cell proliferation. The inhibitory effects of PGE2 were mimicked by the addition of dibutyryl-cyclic AMP, and the effectiveness of PGE2 was markedly potentiated by 1-methyl-3-isobutylxanthine, a potent inhibitor of cyclic nucleotide phosphodiesterase activity. PGE2 caused a 10-fold elevation of the intracellular cyclic AMP concentration, whereas CSF-1 neither increased cyclic AMP levels nor attenuated the rise in cyclic AMP promoted by PGE2. However, CSF-1 may indirectly regulate cyclic AMP levels since in the absence of CSF-1, BAC1.2F5 cells actively synthesized PGE2, whereas PGE2 production was abruptly terminated by the addition of CSF-1. In BAC1.2F5 cells, PGE2 increases the intracellular cyclic AMP concentration, thereby blocking cell proliferation, but does not down-regulate the CSF-1 receptor or abrogate the functions of CSF-1 necessary for cell survival.