Biomedical subjects

M F Roussel

Publications and source records attributed to M F Roussel.

106 records · Page 6Linked to original sources

PubMed1987

Multilineage hematopoietic disorders induced by transplantation of bone marrow cells expressing the v-fms oncogene.

Mouse bone marrow cells infected with a helper-free retrovirus containing v-fms were engrafted into lethally irradiated mice. Dominant provirus-positive clones emerged in the spleens of some recipients within 1 month. When spleen cells were transplanted into lethally irradiated secondary recipients, clonal erythroleukemias or B cell lymphomas expressing the v-fms-coded glycoprotein developed. Other secondary recipients repopulated by "unmarked" progenitor cells or by cryptic provirus-positive precursors present in the spleens of the same donor mice did not develop disease; thus cells expressing v-fms did not invariably have a proliferative advantage after transplantation. Several primary engrafted recipients developed myeloproliferative disorders that were provirus-positive without evidence of clonality. Although expression of the c-fms product (CSF-1 receptor) is normally restricted to cells of the mononuclear phagocyte series, the v-fms-coded glycoprotein can contribute to proliferative abnormalities of multiple hematopoietic lineages.

Animals↗

PubMed1987

Transformation by the v-fms oncogene product: an analog of the CSF-1 receptor.

The product of the c-fms proto-oncogene is related to, and possibly identical with, the receptor for the macrophage colony-stimulating factor, M-CSF (CSF-1). Unlike the product of the v-erbB oncogene, which is a truncated version of the EGF receptor, the glycoprotein encoded by the v-fms oncogene retains an intact extracellular ligand-binding domain so that cells transformed by v-fms express CSF-1 receptors at their surface. Although fibroblasts susceptible to transformation by v-fms generally produce CSF-1, v-fms-mediated transformation does not depend on an exogenous source of the growth factor, and neutralizing antibodies to CSF-1 do not affect the transformed phenotype. An alteration of the v-fms gene product at its extreme carboxyl-terminus represents the major structural difference between it and the c-fms-coded glycoprotein and may affect the tyrosine kinase activity of the v-fms-coded receptor. Consistent with this interpretation, tyrosine phosphorylation of the v-fms products in membranes was observed in the absence of CSF-1 and was not enhanced by addition of the murine growth factor. Cells transformed by v-fms have a constitutively elevated specific activity of a guanine nucleotide-dependent, phosphatidylinositol-4,5-diphosphate-specific phospholipase C. We speculate that the tyrosine kinase activity of the v-fms/c-fms gene products may be coupled to this phospholipase C, possibly through a G regulatory protein, thereby increasing phosphatidylinositol turnover and generating the intracellular second messengers diacylglycerol and inositol triphosphate.

Animals↗

PubMed1987

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.

Animals↗

PubMed1986

Antibodies to distal carboxyl terminal epitopes in the v-fms-coded glycoprotein do not cross-react with the c-fms gene product.

The product of the v-fms oncogene is an integral transmembrane glycoprotein that is closely related to the cell surface receptor for the macrophage colony stimulating factor, CSF-1. A fragment of the v-fms gene encoding a major portion of the extracellular amino terminal domain, the membrane-spanning segment, and the entire carboxyl terminal tyrosine kinase domain of the glycoprotein was molecularly cloned into an inducible prokaryotic expression plasmid. Polypeptide products consisting only of v-fms-coded amino acids were produced in bacteria and were used to prepare immune reagents that precipitated the v-fms-coded glycoproteins expressed in transformed cells. Whereas rabbit antisera to recombinant polypeptides detected antigenic determinants of the c-fms proto-oncogene product, seven mouse monoclonal antibodies to these same antigens reacted only with v-fms-specific epitopes. Proteolytic mapping experiments and studies with a mutant v-fms-coded glycoprotein lacking the 37 carboxyl terminal amino acids of the wild-type product showed that the monoclonal antibodies were restricted in their reactivity to epitopes at the extreme carboxyl terminus of the glycoprotein. The v-fms and c-fms gene products must differ significantly in this region.

Amino Acid Sequence↗

PubMed1986

The amino-terminal domain of the v-fms oncogene product includes a functional signal peptide that directs synthesis of a transforming glycoprotein in the absence of feline leukemia virus gag sequences.

The nucleotide sequence of a 5' segment of the human genomic c-fms proto-oncogene suggested that recombination between feline leukemia virus and feline c-fms sequences might have occurred in a region encoding the 5' untranslated portion of c-fms mRNA. The polyprotein precursor gP180gag-fms encoded by the McDonough strain of feline sarcoma virus was therefore predicted to contain 34 v-fms-coded amino acids derived from sequences of the c-fms gene that are not ordinarily translated from the proto-oncogene mRNA. The (gP180gag-fms) polyprotein was cotranslationally cleaved near the gag-fms junction to remove its gag gene-coded portion. Determination of the amino-terminal sequence of the resulting v-fms-coded glycoprotein, gp120v-fms, showed that the site of proteolysis corresponded to a predicted signal peptidase cleavage site within the c-fms gene product. Together, these analyses suggested that the linked gag sequences may not be necessary for expression of a biologically active v-fms gene product. The gag-fms sequences of feline sarcoma virus strain McDonough and the v-fms sequences alone were inserted into a murine retroviral vector containing a neomycin resistance gene. Both constructs were biologically active when transfected into NIH 3T3 cells and produced morphologically transformed foci at equivalent efficiencies. When transfected into a cell line (psi 2) expressing complementary viral gene functions, G418-resistant (Neor) cells containing either of these vector DNAs produced high titers of transforming viruses. Analysis of proteins produced in cells containing the vector lacking gag gene sequences showed that gP180gag-fms was not synthesized, whereas normal levels of both immature gp120v-fms and mature gp140v-fms were detected. The glycoprotein was efficiently transported to the cell surface, and it retained wild-type tyrosine kinase activity. We conclude that a cryptic hydrophobic signal peptide sequence in v-fms was unmasked by gag deletion, thereby allowing the correct orientation and transport of the v-fms gene product within membranous organelles. It seems likely that the proteolytic cleavage of gP180gag-fms is mediated by signal peptidase and that the amino termini of gp140v-fms and the c-fms gene product are identical.

Amino Acid Sequence↗

PubMed1986

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.

Animals↗

PubMed1986

Molecular cloning, expression, and chromosomal localization of the gene encoding a human myeloid membrane antigen (gp150).

DNA from a tertiary mouse cell transformant containing amplified human sequences encoding a human myeloid membrane glycoprotein, gp150, was used to construct a bacteriophage lambda library. A single recombinant phage containing 12 kilobases (kb) of human DNA was isolated, and molecular subclones were then used to isolate the complete gp150 gene from a human placental genomic DNA library. The intact gp150 gene, assembled from three recombinant phages, proved to be biologically active when transfected into NIH 3T3 cells. Molecular probes from the gp150 locus annealed with a 4.0-kb polyadenylated RNA transcript derived from human myeloid cell lines and from tertiary mouse cell transformants. The gp150 gene was assigned to human chromosome 15, and was subchromosomally localized to bands q25-26 by in situ hybridization. The chromosomal location of the gp150 gene coincides cytogenetically with the region assigned to the c-fes proto-oncogene, another human gene specifically expressed by myeloid cells.

Antigens, Surface↗

PubMed1986

Requirements for transformation by the fms oncogene product (CSF-1 receptor).

The c-fms proto-oncogene product is a transmembrane glycoprotein that is probably identical to the cell surface receptor for the mononuclear phagocyte colony stimulating factor, CSF-1. An analogous glycoprotein encoded by the viral oncogene v-fms includes the extracellular ligand-binding domain, membrane spanning segment, and cytoplasmic tyrosine kinase domain of the CSF-1 receptor. The v-fms and c-fms gene products differ significantly at their distal carboxylterminal ends where the truncated viral transforming protein has lost a single tyrosine residue (tyr969) that may negatively regulate the receptor kinase activity. Introduction of v-fms into a CSF-1 dependent murine macrophage cell line induced factor independence and tumorigenicity by a nonautocrine mechanism. Thus, although the v-fms gene product can bind CSF-1, its constitutive tyrosine-specific protein kinase provides growth stimulatory signals in the absence of ligand. Transfection of human c-fms cDNA into mouse NIH-3T3 cells conferred a CSF-1 responsive phenotype. Although neither the wild-type c-fms (tyr969) gene nor a mutant c-fms (phe969) allele induced transformation of NIH-3T3 cells, cotransfection with human CSF-1 cDNA gave rise to transformed foci. In cells cotransfected with the CSF-1 gene, the efficiency of focus formation induced by the mutant c-fms (phe969) gene was greater than that of the wild-type gene and equivalent to that of v-fms alone. A chimeric v-fms/c-fms molecule in which the carboxylterminus of the v-fms gene product was replaced by the corresponding region of the wild type c-fms (tyr969) was weakly transforming, whereas chimeric molecules containing phe969 transformed NIH-3T3 cells efficiently. Thus, complete oncogenic activation of the c-fms gene appears to require two events: one which alters a putative negative regulatory site of tyrosine phosphorylation, and a second which phenocopies a ligand-induced conformational change.

Animals↗

PubMed1985

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.

Animals↗

PubMed1985

Transmembrane orientation of glycoproteins encoded by the v-fms oncogene.

The retroviral oncogene v-fms encodes a glycoprotein whose transport to the plasma membrane is required for transformation. Tryptic digestion of microsomes from transformed cells yielded membrane-protected amino-terminal fragments 40 kd smaller than intact molecules. These fragments were glycosylated, and they included v-fms-coded epitopes expressed at the cell surface. Deletion of the predicted membrane-spanning peptide generated polypeptides that were completely sequestered within microsomes. The mutant glycoproteins acquired more asparagine-linked oligosaccharide chains than did wild-type molecules, lacked kinase activity in vitro, were not transported to the cell surface, and had no transforming activity. Thus, the membrane-spanning segment in the middle of the glycoprotein interrupts translocation of nascent chains into the endoplasmic reticulum, ultimately orienting the amino-terminal domain outside the cell and the carboxy-terminal kinase domain in the cytoplasm. These topological features are similar to those of several growth factor receptors, suggesting that v-fms transforms cells through modified receptor-mediated signals.

Amino Acid Sequence↗

PubMed1985

The c-fms proto-oncogene product is related to the receptor for the mononuclear phagocyte growth factor, CSF-1.

The feline c-fms proto-oncogene product is a 170 kd glycoprotein with associated tyrosine kinase activity. This glycoprotein was expressed on mature cat macrophages from peritoneal inflammatory exudates and spleen. Similarly, the receptor for the murine colony-stimulating factor, CSF-1, is restricted to cells of the mononuclear phagocytic lineage and is a 165 kd glycoprotein with an associated tyrosine kinase. Rabbit antisera to a recombinant v-fms-coded polypeptide precipitated the feline c-fms product and specifically cross-reacted with a 165 kd glycoprotein from mouse macrophages. This putative product of the murine c-fms gene exhibited an associated tyrosine kinase activity in immune complexes, specifically bound murine CSF-1, and, in the presence of the growth factor, was phosphorylated on tyrosine in membrane preparations. The murine c-fms proto-oncogene product and the CSF-1 receptor are therefore related, and possibly identical, molecules.

Amino Acid Sequence↗

PubMed1985

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.

Animals↗

PubMed1984

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.

Animals↗

PubMed1983

Molecular cloning of the c-fms locus and its assignment to human chromosome 5.

Molecular clones of the retroviral oncogene v-fms were used to isolate recombinant bacteriophages containing c-fms proto-oncogene sequences from a human placental DNA library. Viral and cellular fms sequences were used in Southern blotting experiments with a panel of 32 human X mouse somatic cell hybrids to assign the human c-fms proto-oncogene to human chromosome 5.

Animals↗

PubMedDate not supplied

Transforming potential of the c-fms proto-oncogene (CSF-1 receptor).

The c-fms proto-oncogene encodes a transmembrane glycoprotein that is probably identical to the receptor for the macrophage colony stimulating factor, CSF-1. Forty C-terminal amino acids of the normal receptor are replaced by 11 unrelated residues in the feline v-fms oncogene product, deleting a C-terminal tyrosine residue (Tyr969) whose phosphorylation might negatively regulate the receptor kinase activity. We show that the human c-fms gene stimulates growth of mouse NIH 3T3 cells in agar in response to human recombinant CSF-1, indicating that receptor transduction is sufficient to induce a CSF-1 responsive phenotype. Although cells transfected with c-fms genes containing either Tyr969 or Phe969 were not transformed, cotransfection of these genes with CSF-1 complementary DNA induced transformation, with c-fms(Phe969) showing significantly more activity than c-fms(Tyr969). In the absence of CSF-1, chimaeric v-fms/c-fms genes encoding the wild-type c-fms C terminus were poorly transforming, whereas chimaeras bearing Phe969 were as transforming as v-fms. Thus, the Phe969 mutation, although not in itself sufficient to induce transformation, activates the oncogenic potential of c-fms in association with an endogenous ligand or in conjunction with mutations elsewhere in the c-fms gene that confer ligand-independent signals for growth.

Amino Acid Sequence↗

PubMedDate not supplied

Synthesis, post-translational processing, and autocrine transforming activity of a carboxylterminal truncated form of colony stimulating factor-1.

The mononuclear phagocyte colony stimulating factor encoded by a 1.6 kilobase pair human cDNA is synthesized as a homodimeric transmembrane glycoprotein that is released from the plasma membrane by proteolysis. Premature termination of the CSF-1 coding sequence upstream of its carboxylterminal transmembrane-spanning segment and expression of the truncated CSF-1 cDNA in either bovine papilloma virus or retrovirus vectors led to the synthesis of a soluble, biologically active growth factor that was rapidly secreted from cells. Like the full-length CSF-1 precursor, the truncated polypeptide was rapidly assembled through disulfide bonds immediately after synthesis and acquired asparagine-linked oligosaccharide chains that underwent progressive post-translational modifications during intracellular transport. Soluble CSF-1 encoded by the truncated cDNA stimulated the formation of bone marrow-derived mouse macrophage colonies in semisolid medium and induced transformation of mouse NIH-3T3 cells when coexpressed with the human c-fms proto-oncogene product (CSF-1 receptor). Compared to results obtained with the full-length CSF-1 cDNA, the efficiency of transformation obtained with the truncated CSF-1 gene was reduced, in spite of the fact that transfected cultures produced similar levels of the extracellular growth factor. The results indicate that CSF-1 amino acid residues 1-158 (together with the aminoterminal signal peptide at residues -32 to -1) are sufficient for biological activity and that CSF-1 cDNAs encoding either membrane-bound or soluble precursors are active in autocrine transformation.

Animals↗