The v-fms oncogene induces factor independence and tumorigenicity in CSF-1 dependent macrophage cell line

The McDonough strain of feline sarcoma virus (SM-FeSV) transforms fibroblast cell lines in culture and produces fibrosarcomas in domestic cats. SM-FeSV does not induce haematopoietic malignancies in spite of the fact that its viral oncogene, v-fms, codes for a glycoprotein related to the receptor for the mononuclear phagocyte colony stimulating factor, CSF-1. The v-fms-coded polypeptide includes the complete extracellular domain of the c-fms proto-oncogene product and retains the ability to bind CSF-1 specifically. The two molecules have very similar sequences except at their extreme carboxyl terminal ends where 40 amino acids of the c-fms-coded glycoprotein are replaced by 11 unrelated residues in the v-fms product. Autophosphorylation of the c-fms gene product on tyrosine is enhanced by CSF-1 addition, whereas phosphorylation of the v-fms-coded glycoprotein appears to be constitutive. We now show that introduction of the v-fms gene into simian virus-40 (SV40)-immortalized, CSF-1 dependent macrophages renders them independent of CSF-1 for growth and tumourigenic in nude mice. These factor-independent cell lines express unaltered levels of the c-fms product which is down-modulated in response to either CSF-1 or the tumour promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The induction of factor independence by a non-autocrine mechanism suggests that the v-fms product is an unregulated kinase that provides growth stimulatory signals in the absence of ligand.     

Structural alteration of viral homologue of receptor proto-oncogene fms at carboxyl terminus

A role for proto-oncogenes in the regulation and modulation of cell proliferation has been suggested by the findings that the B-chain of platelet-derived growth factor (PDGF) is encoded by the proto-oncogene sis and that the erb-B oncogene product is a truncated form of the epidermal growth factor (EGF) receptor. Furthermore, the product of the proto-oncogene fms (c-fms) may be related or identical to the receptor for macrophage colony-stimulating factor (CSF-1). v-fms is the transforming gene of the McDonough strain of feline sarcoma virus (SM-FeSV) and belongs to the family of src-related oncogenes which have tyrosine-specific kinase activity. Furthermore, nucleotide sequence analysis of the v-fms gene product revealed topological properties of a cell-surface receptor protein. To elucidate the features involved in the conversion of a normal cell-surface receptor gene into an oncogenic one, we have now determined the complete nucleotide sequence of a human c-fms complementary DNA. The 972-amino-acid c-fms protein has an extracellular domain, a membrane-spanning region, and a cytoplasmic tyrosine protein kinase domain. Comparison of the feline v-fms and human c-fms sequences reveals that the proteins share extensive homology but have different carboxyl termini.     

Apparent role of the macrophage growth factor, CSF-1, in placental development

Colony stimulating factor-1 (CSF-1) is a glycoprotein growth factor required for the proliferation and differentiation of mononuclear phagocytic cells (reviewed in ref. 1). A 10,000-fold elevation of mouse uterine CSF-1 during pregnancy, suggested by studies of the bone marrow colony stimulating activity of uterine extracts, was recently demonstrated by radioimmunoassay (RIA). This increase and the observations that placenta and choriocarcinoma cell lines express c-fms messenger RNA and the c-fms proto oncogene product (CSF-1 receptor) respectively, suggest an additional role for CSF-1 in pregnancy. We now show that uterine CSF-1 concentration is regulated by the synergistic action of female sex steroids, oestradiol-17 beta (E2) and progesterone (P) and that the elevation in CSF-1 concentration can be attributed to the preferential expression of an alternatively spliced CSF-1 mRNA by uterine glandular epithelial cells. These findings indicate that CSF-1, under hormonal influence, plays a role in placental development and function and that steroid hormones may regulate developmental processes via their effects on the expression of tissue-specific growth factors.     

Myc rescue of a mutant CSF-1 receptor impaired in mitogenic signalling.

The colony-stimulating factor-1 receptor (CSF-1R) mediates its pleiotropic effects through the coupling of its ligand-activated tyrosine kinase to multiple intracellular effector proteins, whose combined actions determine the magnitude and specificity of the biological response. The interaction of cytoplasmic signalling molecules with CSF-1R is mediated in part by sequence motifs flanking sites of receptor tyrosine phosphorylation. Mutation of an autophosphorylation site at tyrosine 809 in the cytoplasmic domain of human CSF-1R does not significantly reduce its ligand-stimulated tyrosine kinase activity, binding to phosphatidylinositol 3-kinase, or induction of the immediate early response genes, c-fos and junB (ref.2). Unlike cells bearing wild-type receptors, mouse NIH3T3 cells expressing mutant CSF-1R(Phe 809) were unable to grow in serum-free medium containing human recombinant CSF-1 and did not form colonies in semi-solid medium in its presence. CSF-1 induction of c-myc messenger RNA in these cells was impaired, but enforced expression of an exogenous c-myc gene restored their ability to proliferate in response to the growth factor. These studies demonstrate a receptor-mediated bifurcation of intracellular signal transduction pathways during the immediate early response and assign a central role for c-myc in CSF-1-induced mitogenesis.     

Frequent c-fms activation by proviral insertion in mouse myeloblastic leukaemias

Retroviruses lacking oncogenes can induce tumours in animals, and the tumour cells are frequently found to contain proviral DNA inserted next to a proto-oncogene, which is thus placed under the regulatory control of the retroviral long terminal repeat (LTR). This altered regulation leads to overexpression of the proto-oncogene, which presumably contributes to the growth properties of the tumour cells. fim-2 has been described as a retroviral integration site frequently and specifically involved in murine myeloblastic leukaemias induced in vivo or in vitro by the replication-competent Friend murine leukaemia virus (F-MuLV). Here we report that fim-2 spans the 5'-end of the murine proto-oncogene c-fms, known to code for a transmembrane glycoprotein with tyrosine kinase activity probably identical to the receptor of the haemopoietic growth factor, monocyte-macrophage colony-stimulating factor (M-CSF or CSF-1). Proviral integration in the fim-2 region results in a high expression of a normal sized c-fms messenger RNA. We also observe that some tumours have lost the fim-2/c-fms germ line allele. These results provide the first evidence for the presumed involvement of c-fms in myelomonocytic leukaemias.     

Signal transduction pathways mediated by colony stimulating factor-1 receptor

Colony-stimulating factor-1 (CSF-1), which is necessary for cell proliferation and differentiation, regulates both immediate and delayed early responses throughout G1 phase. The binding of CSF-1 to its receptor (CSF-1R) triggers phosphorylation of the receptor and its intrinsic tyrosine kinase. The activated receptor binds directly to cytoplasmic effector proteins, which induce multiple-signal transduction pathways. CSF-1 can induce the c-myc gene expression via Ras and Ets-related proteins. The expression of c-fos/jun family genes is also targeted following the activation of Ras. CSF-1R activates STAT1 and STAT3 to participate in signaling, but JAKs do not appear to contribute to signaling by CSF-1R. CSF-1R activates PI3-kinase, and PI3-ki can interact with downstream proteins by the MAPKK-related pathway independent of Ras/Raf. PC-PLC can enforce signaling in response to CSF-1. Furthermore, the turnover and dephosphorylation by the phosphatase SHPTP1 of CSF-1R are the major mechanism in the negative regulation of signaling by CSF-1R     

The colony stimulating factor-1 receptor associates with and activates phosphatidylinositol-3 kinase

Colony stimulating factor-1 (CSF-1) is a lineage-specific growth factor required for proliferation and survival of mononuclear phagocytes and their precursors. The CSF-1 receptor belongs to a family of ligand-activated protein-tyrosine kinases. Activation of the platelet-derived growth factor receptor, but not the CSF-1 receptor, leads to an increase in phospholipase C activity and a subsequent elevation in intracellular calcium. Recent studies have shown that a novel phosphoinositol (PtdIns) kinase, termed PtdIns-3 kinase, is stimulated by the platelet-derived growth factor receptor and certain oncogenes in the protein-tyrosine kinase family. PtdIns-3 kinase phosphorylates the D-3 hydroxyl position of the inositol ring of PtdIns, and its products do not participate in the generation of the second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Here we report that addition of CSF-1 is followed by activation of PtdIns-3 kinase in a macrophage cell line (P388 D1), which contains CSF-1 receptors, and in BALB/c fibroblasts made to express the human CSF-1 receptor. Furthermore, we show that activation of the CSF-1 receptor results in the accumulation in intact cells of polyphosphoinositides phosphorylated at the D-3 position of the inositol ring. Thus activation of the CSF-1 receptor stimulates PtdIns-3 kinase activity, indicating a novel pathway for CSF-1 receptor-mediated signal transduction.     

An artefact explains the apparent association of the transferrin receptor with a ras gene product

There is substantial evidence implicating ras genes in a number of human neoplasms. The ras genes of several human tumours display mutational changes which are likely to be responsible for their transforming activity. Normal cells also express ras genes, over-expression of which can induce cellular transformation. ras genes encode proteins of approximately 21,000 molecular weight (MW) (p21) that are localized to the inner surface of the plasma membrane. Much effort is being focused on the elucidation of the physiological function of ras-encoded proteins in normal and transformed cells, concentrating on interactions between p21 and other cellular elements. Recently, Finkel and Cooper reported that p21 in extracts of human bladder carcinoma cells is involved in a molecular complex with the transferrin receptor of these cells. This report aroused considerable interest, particularly as expression of the transferrin receptor has been linked to cell proliferation. I present here evidence that the apparent association of p21 and the transferrin receptor is an artefact of the immunoprecipitation technique.     

Human transforming growth factors induce tyrosine phosphorylation of EGF receptors

Cultured cell lines of human tumour origin as well as cells transformed by various RNA tumour viruses secrete low molecular weight polypeptide transforming growth factors (TGFs). In addition to competing with epidermal growth factor (EGF) for binding to its cellular receptor, TGFs can transform morphologically fibroblast and epithelial cells in culture. In view of accumulating evidence that tyrosine phosphorylation activity is associated with the transforming genes of various tumour viruses, we determined whether phosphotyrosine levels were elevated in these human tumour cells. We show here that TGFs produced by human tumour cells induce phosphorylation of specific tyrosine acceptor sites in the 160,000-molecular weight (160 K) EGF receptor.     

Amplification, enhanced expression and possible rearrangement of EGF receptor gene in primary human brain tumours of glial origin

Epidermal growth factor (EGF), through interaction with specific cell surface receptors, generates a pleiotropic response that, by a poorly defined mechanism, can induce proliferation of target cells. Subversion of the EGF mitogenic signal through expression of a truncated receptor may be involved in transformation by the avian erythroblastosis virus (AEV) oncogene v-erb-B, suggesting that similar EGF receptor defects may be found in human neoplasias. Overexpression of EGF receptors has been reported on the epidermoid carcinoma cell line A431, in various primary brain tumours and in squamous carcinomas. In A431 cells the receptor gene is amplified. Here we show that 4 of 10 primary brain tumours of glial origin which express levels of EGF receptors that are higher than normal also have amplified EGF receptor genes. Amplified receptor genes were not detected in the other brain tumours examined. Further analysis of EGF receptor defects may show that such altered expression and amplification is a particular feature of certain human tumours.     

Structure of the receptor for platelet-derived growth factor helps define a family of closely related growth factor receptors

The primary structure of the receptor for platelet-derived growth factor (PDGF), determined by means of cloning a cDNA that encodes the murine pre-PDGF receptor, is closely related to that of the v-kit oncogene product and the receptor for macrophage colony stimulating factor (CSF-1). Common structural features include the presence of long sequences that interrupt the tyrosine-specific protein kinase domains of each molecule. The PDGF and CSF-1 receptors also share a characteristic distribution of extracellular cysteine residues. Ubiquitin is covalently bound to the purified PDGF receptor, the human gene for which is on chromosome 5.     

Cell transformation and activation of pp60c-src by overexpression of a protein tyrosine phosphatase.

The kinase activity of pp60c-src is specifically and transiently increased during mitosis and repressed during interphase. Loss of cell-cycle control of pp60c-src occurs on mutation of Tyr527 to Phe or when pp60c-src is associated with polyoma middle-T-antigen, and these conditions result in cell transformation or tumorigenesis. In both cases, pp60c-src has elevated kinase activity which is maintained throughout the cell cycle and accompanied by dephosphorylation of the carboxy-terminal negative regulatory Tyr527 site, or mimicry of Tyr527 dephosphorylation in the case of the mutant. Here we report that overexpression of the receptor-like protein tyrosine phosphatase PTP alpha results in persistent activation of pp60c-src kinase, with concomitant cell transformation and tumorigenesis. In PTP alpha-overexpressing cells, the pp60c-src kinase activation is accompanied by dephosphorylation at Tyr527, and direct dephosphorylation of this site by purified PTP alpha occurs in vitro. Our results suggest that PTP alpha is involved in the regulation of cell proliferation, exerting at least some of its effects through pp60c-src kinase, and has oncogenic capability when overexpressed.     

Focus formation in rat fibroblasts exposed to a tumour promoter after transfer of polyoma plt and myc oncogenes

The gene encoding the large-T protein of polyoma virus (plt), the E1A genes of adenoviruses, the viral myc gene (v-myc) or rearranged forms of the cellular c-myc gene confer on rat embryo fibroblast (REF) cells in primary culture a series of new properties ('immortality', reduced serum requirement and sensitivity to transformation by viral and activated cellular oncogenes) but do not induce the appearance of transformed foci. We now report that focus formation can be induced after transfer of these genes into either REF or established FR3T3 rat cells by subsequent exposure to the tumour promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). Frequencies of transformation are in the same range as those usually observed for transformation with complete polyoma DNA or with a mixture of cloned myc and ras oncogenes. These results further characterize the 'immortalized' state induced by plt and myc as one in which the cells maintain a normal growth control in many respects but can be further acted upon to produce a neoplastic progeny.     

Requirement for c-ras proteins during viral oncogene transformation

Many retroviral oncogenes have been classified into one of several categories based on structure, enzymology and cellular localization. These genes originated from host cells and are probably derived from genes normally involved in the control of cell proliferation. The cellular counterparts of three oncogenes have been identified as a growth factor or growth factor receptor; related oncogenes include receptor-like membrane proteins which often express tyrosine kinase activity. These growth factor-related oncogenes are structurally and biochemically distinct from the membrane-associated ras gene family, which bind and hydrolyse GTP. Oncogenes localized primarily in the cytoplasm which probably have serine kinase activity, have also been identified. Although the structure and biochemistry of many oncogenes have been extensively studied, relatively little is known about the functional relationships of oncogene proteins within the cell. An opportunity to study such interaction is provided by the identification of a monoclonal antibody that neutralizes cellular ras proteins when microinjected into cells. It has been shown previously that the injected antibody inhibits the initiation of S-phase in NIH 3T3 cells. In the present study we injected this monoclonal antibody into NIH 3T3 cells transformed by a variety of oncogenes. The results show that transformation by three growth factor receptor-like oncogenes depends on c-ras proteins, while transformation by two cytoplasmic oncogenes appears to be independent of c-ras protein.     

Influence of site-directed mutation of cytochrome b5 Phe35 on the protein’s structure and properties

Kinetic studies of the heme dissociation from the wild type and Phe35Tyr, Phe35Leu mutants of bovine liver microsomal ferricytochrome b 5 indicate that the oxidized Phe35Tyr mutant is more stable towards denaturant than wild type but Phe35Leu mutant proceeded with a different mechanism compared with wild type cytochrome b 5 and Phe35Tyr mutant protein. Because of the decrease of side chain volume in Phe35Leu mutant, a cavity produced in the interior of the protein may offer a channel for urea molecule to enter the hydrophobic pocket. When urea concentration is larger than 5 mol/L, the urea molecule may compete to coordinate the iron of heme with His39, that results in sharp increase of the rate of heme dissociation. The interaction between cytochrome b 5 and cytochrom c demonstrated that a 1:1 protein complex was formed between the two proteins. The binding constants of cytochrome b 5 with cytochrome c are: wild type K A=4.2(±0.01)×10 6(mol/L) -1 , Phe35Tyr K A=3.7(±0.01)×10 6(mol/L) -1 and Phe35Leu K A=4.7(±0.01)×10 6(mol/L) -1 respectively ( I =1 m mol/L, pH 7.0 soldium phosphate buffer, 25℃). These results clearly show that the mutation at Phe35 has no influence on the binding of cytochrome b 5 with cytochrome c and that the hydrophilic patch residues are not involved in the binding of cytochrome b 5 and cytochrome c.     

Transmission of the polyoma virus middle T gene as the oncogene of a murine retrovirus

Polyoma virus is a papovavirus that productively infects mouse cells. In cells of other species, such as rat cells, polyoma virus is virtually unable to replicate, and a small proportion of infected cells become stably transformed. The ability of polyoma virus to transform infected cells is determined by genes that encode the large, middle and small T antigens and which are found in the early region of the virus genome. We have inserted the transforming region of polyoma virus into a murine leukaemia virus (MLV) vector, to generate a replication-defective transforming retrovirus which for the first time allows efficient transformation of mouse cells by the polyoma virus middle T gene. During the life cycle of this recombinant virus the intervening sequence present in the original polyoma virus middle T gene was removed. The recombinant virus that we have constructed is analogous to other acutely transforming retroviruses, and demonstrates that the polyoma middle T gene is a dominant transforming oncogene.     

Suppression of c-ras transformation by GTPase-activating protein

The ras genes are required for normal cell growth and mediate transformation by oncogenes encoding protein tyrosine kinases. Normal ras can transform cells in vitro and in vivo, but mutationally activated ras does so much more efficiently, and highly transforming mutant versions of ras have been isolated from a variety of human and animal tumours. The ras genes encode membrane-associated, guanine nucleotide-binding proteins that are active when GTP is bound and inactive when GDP is bound. The slow intrinsic GTPase activity of normal mammalian Ras proteins can be greatly accelerated by the GTPase-activating protein (GAP), which is predominantly cytoplasmic. This activity of GAP, which can increase with cell density in contact-inhibited cells, suggests that it functions as a negative, upstream regulator of ras. Other studies, however, show that GAP interacts with a region of ras-encoded protein implicated in ras effector function, which raises the possibility that GAP might also be a downstream target of ras. Mutationally activated ras-encoded proteins also interact with GAP, although they are resistant to its catalytic activity. In an attempt to define the role of GAP in ras-mediated transformation, we examined the effects on transformation of normal or mutant ras when cells overexpress GAP. We found that GAP suppresses transformation of NIH 3T3 cells by normal Ha-ras (c-ras) but does not inhibit transformation by activated Ha-ras (v-ras). These results support the hypothesis that GAP functions as a negative regulator of normal ras and make it unlikely that GAP alone is the ras target.