International Journal of Cell Cloning 8:46-62Suppl.1 (1990)

Regulation of Mononuclear Phagocyte Proliferation by Colony-Stimulating Factor-1 Charles J. Sherr Howard Hughes Medical Institute, Department of Tumor Cell Biology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA

Key Words. Colony-stimulatingfactor-1 (CSF-1) CSF-1 receptor c-fms protooncogene Mononuclear phagocytes Cell transformation Leukemia Abstract. Colony-stimulating factor-1 (CSF-1 or M-CSF) regulates pleiotropic developmental and functional responses of macrophages and their committed bone marrow progenitors and supports the viability of cells of the mononuclear phagayte lineage. Its actions are mediated through its binding to cell surface CSF-I receptors (CSF-lR) that exhibit ligand-stimulated tyrosine kinase activity. CSF-lR-induced phosphorylation of intracellular protein substrates initiates a cascade of biochemical reactions that relay signals to the cell nucleus, elicit transcription of CSF-I-responsivegenes and culminatein cell division. The actions of the CSF-lR kinase can be interrupted by binding of certain monoclonal antibodies to the extracellular domain of the receptor or by agents which activate protein kinase C and accelerate receptor turnover. CSF-LR is encoded by the c-fms proto-oncogene, and specific genetic alterations, which constitutivelyactivate the receptor kinase, provide sustained signals for cell growth leading to cell transformation.Perturbations in the structure or expression of the c-fms proto-oncogene might therefore contribute to leukemia.

Introduction Colony-stimulatingfactor-I (CSF-1 or M-CSF) is a lineage-specificgrowth factor that stimulates the proliferation, differentiationand survival of cells of the mononuclear phagocyte series [l, 21. It is distinguished by its specificity of action and physicochernical properties from granulocyte-macrophage(GM) CSF and interleukin 3 (IL-3), both of which stimulate more immature myeloid cells, including precursors of monocytes and macrophages [3]. All of the actions of CSF-1 are mediated through its binding to specific cell surface receptors encoded by the c-fms proto-oncogene [4]. The CSF-1 receptor (CSF-lR) exhibits a liganddependentprotein-tyrosine kinase activity,providing a relatively well-characterized system for dissecting the function of such enzymes in signal transduction in hematopoietic cells. Correspondence: Charles 1. Sherr, M.D., Ph.D., Howard Hughes Medical Institute, Department of Tumor Cell Biology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105, USA. Received September 7, 1989; accepted for publication September 7, 1989. 0737-1454/90/$2.0010 @AlphaMedPress

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CSF-1Structure, Biosynthesis and Action The original biologic assay for CSF-1 activity, which involves factordependent colony formation by single bone marrow precursors plated in semisolid medium [5,6], was used to purify the growth factor to homogeneity [7]. This enabled development of sensitive radioimmuno-and radioreceptor assays for specific quantitation of CSF-1 in serum, urine or cell culture medium [8]. Because purified CSF-1 is relatively stable and can be radioiodinated to high-specific activity, the latter assays can detect picomolar concentrationsof the growth factor that are well within the range necessary to elicit a biological response. Determination of the aminoterminal amino acid sequence of purified CSF-1 allowed the construction of oligonucleotideprobes that were used to clone the gene [9]. Molecular cloning and characterizationof human and mouse CSF-1 c D N h [9-141 and their expression in eukaryotic systems has facilitated production of purified CSF-1 in quantities that were formerly unobtainable from biologic fluids, thereby yielding sufficient quantities for whole animal experiments and future clinical trials. The human CSF-1 gene has been assigned to the long arm of chromosome 5 at band q33.1 [l5-17] and specifies a group of differentially spliced RNAs that encode different forms of the growth factor [9-141. A 4 Kb mRNA specifies a 554 amino acid precursor (CSF-lSs4)which is synthesized as an integral transmembrane glycoprotein. This precursor is glycosylated,dimerized and proteolytically cleaved in the intracellular secretory compartment to yield a soluble 90 Kd homodimeric glycoprotein that is then secreted from the cell [18, 191. Smaller spliced transcripts of 1.8 to 2.3 Kb differ from the 4 Kb mRNA species in their 3' nontranslatedregions, but encode an identical protein product. The one exception is a 1.6 Kb transcript, originally identified and cloned from a pancreatic carcinoma cell line [9], which encodes a transmembrane polypeptide precursor of only 256 amino acids (CSF-1256).Because CSF-12s6lacks recognition sites for the intracellular protease that processes CSF-Ps4,it ultimately generates a plasma membrane-bound form of CSF-1 which retains biologic activity [20]. Although the 1.6 Kb transcript has not been found to be expressed in many CSF-1-producing cells, its identification and pattern of processing suggest that CSF-1 may not only function as a soluble growth factor but may also play a role in direct cell-to-cell interactions. CSF-1 is produced by fibroblasts, including bone marrow stromal cells, and is released into the circulation where it binds to receptors on monocytes, serving to maintain their viability [21]. Although many fibroblast cell lines in tissue culture produce the growth factor, its synthesis may be regulated, at least in part, by other serum growth factors, such as the plateletderivedgrowth factor (PDGF) . Production of CSF-1 by mesenchymal cells appears to account for the constitutive levels of the growth factor normally detected in serum. Because monocytes continually bind, internalizeand degrade the growth factor, their number is homeostatically regulated by this mechanism [21].

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Synthesis of CSF-1 is induced in many cell types in response to other cytokines. Monocytes themselves produce the growth factor after treatment by phorbol esters, interferon-y, GM-CSF and the a-isoform of tumor necrosis factor (TNF-a)[22-251, suggestingthat the release of CSF-1 from activated macrophages may enhance mononuclear phagocyte proliferation during an inflammatory response or at sites of tissue injury (see below). Similarly, CSF-1 is elaborated both by lectin-stimulated T and B cells [26, 271 and by endothelial cells exposed to L-1, TNF or bacterial lipopolysaccharide(LPS)[28]. In contrast to its roles in hematopoiesis and inflammation, CSF-1 is also produced by uterine epithelium during pregnancy where its local level increases almost 1,OOO fold during gestation and is highest at parturition [29]. The expression of functional CSF-1 receptors on placental trophoblasts suggests that, during development, CSF-1 acts as a placental growth factor [30-321. These findings underscore a redundancy in utilizing the same ligand-receptor combination in different physiologic contexts and exemplify a natural case where the CSF-lR kinase can function as a signal transducer outside the context of hematopoiesis. Apart from its role in stimulating cell growth, CSF-1 has been reported to potentiate effector functions of mature mononuclear phagocytes. These include enhancement of phagocytic and cytotoxic activity [33-351; release of prostaglandins, biocidal oxygen metabolites and plasminogen activator [36-391; and production of other cytokines, including IL-1, TNF, interferon and granulocyte(G) CSF 140-421. Because many of the latter factors regulate CSF-1 synthesisand vice versa (see above), it is unclear whether each of the observed effects is an immediate consequence of CSF-1 action. Further complexity in interpretation arises from the dependence of monocytes and macrophages on CSF-1 for their survival.

CSF-lR Structure The CSF-1 receptor is encoded by the c-fmr proto-oncogene [4] and is a member of a family of growth factor receptors that exhibit ligand-stimulated tyrosine b a s e activity [43]. The nucleotide sequence of@ genes from different species [44-471 predicted that the receptor is organized into an aminoterminal extracellular domain containing the ligand-binding site, a single membrane-spanning region, a cytoplasmic tyrosine kinase domain and a short carboxylterminal tail. The extracellular domain consists of five tandem disulfide-bonded loops, each representing motifs characteristic of the immunoglobulin gene superfamily [48]. This region of the polypeptide is extensively glycosylated, and the presence of 11 asparagine-linked oligosaccharidechains contributes approximately 45 Kd to the apparent molecular mass of the ca. 150 Kd receptor. Sequences in the intracellular portion of CSF-lR are evolutionarilyconserved among all protein tyrosine b a s e s , including other receptor kinases and protooncogene products of the src, ubl andfesl’s gene families [43]. This region contains a characteristic gly-X-gly-X-X-gly motif followed by a downstream lysine

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residue which is the site of ATP binding. Although the majority of sequences in the tyrosine kina= domain are homologous to those in other family members, CSF-lR contains a 72 amino acid “spacer” or “kinase insert” region in the middle of its enzymatic domain. The spacer region shows no amino acid similarity to sequences found in other tyrosine kinases, including those family members which entirely lack these sequences, as well as certain other receptor kinases which have analogous spacers of different sequence and length. The distal carboxylterminus of CSF-lR is composed of a short “tail” that has been implicated to negatively regulate tyrosine kinase activity. Disruption of this region by truncation or mutation has been shown to enhance the receptor-mediated CSF-1 response [49,50]. Among the known growth factor receptors, CSF-lR and the B-type receptor for PDGF (PDGF-RB) exhibit amino acid similarity in both their intracellular and extracellular domains [51]. Although the homology is most marked within their kinase domains, the ligand-binding portions of both receptors consist of disulfide-stabilized, immunoglobulin-likeloops, with an identical spacing of cysteine residues throughout. Although the human genes encoding CSF-lR and PDGF-RB were initially mapped to a similar region of chromosome 5 at bands q33.3 and q31-32 [16,17,51], suggesting that they were at least one megabase apart, the two genes were recently shown to be organized in a head-to-tail tandem array [52]. The similarity of the human CSF-lR and PDGF-RB cDNA sequences [45, 511 and knowledge of the intron-exon organization of the human c - j h gene [53] allowed correct predictions of splice junctions within the PDGF-RB gene [52]. Thus, CSF-lR and PDGF-RB were derived by duplication of a single ancestral sequence that underwent subsequent evolutionary divergence. Based on predicted amino acid sequence homology, other members of the CSF-WPDGF-RB subfamily includethe A-type receptor for PDGF [54,55], the receptor for basic fibroblast growth factor [56] and the c-kit proto-oncogene product [57], a putative receptor for an as yet uncharacterized ligand. The cytogenetic assignments of c-kit and the PDGF-R, gene to human chromosome 4 [54,57] suggest that they may also be tandemly linked.

Signal Transduction by CSF-lR CSF-1 binding to its receptor activates the receptor kinase and triggers a cascade of biochemical events that induce cell proliferation. The initiating events involve the phosphorylation of heterologousprotein substrates on tyrosine, at least some of which must function to relay signals that alter the transcription of CSF-1responsive genes. The biochemical steps that link phosphorylation of physiologic substrates with gene activation remain unclear, but must ultimately involve the participationof m - a c t i n g nuclear factorsthat are modulated in response to CSF-1 stimulation. A major challenge is to identify physiologic substrates of the receptor kinase that productively initiate signal transduction along the “mitogenic pathway.”

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Phosphorylation of a series of protein substrates has been observed within seconds of CSF-1 stimulation of receptor-bearing cells, although none of these has yet been characterized in sufficient detail [58-601. CSF-1 can elicit a rapid, pleiotropic response involving membrane ruffling and increased pinaytosis [61], increased sugar transport [62], cytoplasmic alkalinization [63] and the induction of “immediate early response” genes, including the proto-oncogene cfos, within minutes of stimulation [64,651. This suggests that multiple physiologic substrates of CSF-lR kinase will be identified. Because CSF-1 is required throughout the G1 phase of the cell cycle to elicit a mitogenic response [66], different substrates may be phosphorylated as the cells progress toward S phase. Moreover, the fact that low concentrations of the growth factor can maintain macrophage viability without stimulating cell growth [66] argues that a subset of CSF-1responsive genes regulate cell metabolism rather than mitogenesis. Recent observations that both PDGF-RBand the epidermal growth factor receptor (EGF-R) phosphorylate phospholipase C-y (PLC-y) on tyrosine after stimulation by their respective ligands suggested that tyrosine kinases might act to generate lipid “second messengers” [67-711. PLC-y cleaves phosphatidylinositol 4,5-bisphosphate (PIP,), yielding soluble inositol 1,4,5-trisphosphate (IPJ, a physiologic mobilizer of intracellular calcium, and 1,2-diacylglycerol(DAG), an activator of protein b a s e C (PKC) [72]. Both IP3 and PKC can regulate signaling pathways that activate certain genes. In spite of its structural similarity to PDGF-R,, however, CSF-lR does not recognize PLC-y as a substrate [73], consistent with observations that CSF-1 does not mobilize soluble inositides in macrophages [74]. Stimulation of fibroblasts with PDGF or transformation by the v-$ns oncogene (see below) has been reported to induce the phosphorylation of the c-mf-1 protooncogene product [75,76], itself a serineheonine kinase [77]. Because genetic alterations in the c-mf gene that constitutively activate its kinase activity induce cell transformation, a reasonable hypothesis is that the c-ruf-coded kinase functions “downstream” of CSF-LR in signal transduction. Another potential physiologic substrate for the CSF-lR kinase is a novel phosphatidylinositol (PI)-3’ kinase of as yet unknown function 178, 791. PDGF-RB mutants from which the receptor spacer domains have been deleted are impaired in their mitogenic activity [80] and fail to productively interact with either the PI-3’ kinase or with the c-rufgene product [76] even though they retain tyrosine kinase activity. It will be important to determine whether the same e f i t s can be observed with CSF-lR in macrophages. Although in adult animals CSF-1R is normally limited in its expression to mononuclear phagocytes, the cloned c-fms gene has been introduced into a variety of different cell types to assess its ability to couple to downstream components of the mitogenic machinery. Because murine CSF-1 does not bind to human CSF-lR with high affinity [81,82], many such experiments have been performed using the human receptor and rodent target cells to avoid the possibility of clos-

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ing an autocrine loop. When introduced into mouse NIH-3T3 or Chinese hamster lung fibroblasts, CSF-lR expression enabled the cells to proliferate in response to human recombinant CSF-1 [50,83]. Indeed, CSF-1 could replace the requirements of mouse NIH-3T3 cells for PDGF and insulin-like growth factor4 (IGF-1), facilitating cell growth in chemically defined medium containing CSF-1 as the only exogenousgrowth factor [MI. Transductionof CSF-lR into IL.3-dependent mouse myeloid FDC-PI cells also reprogrammedtheir growth factor requirements and enabled the cells to proliferate in CSF-1 [85]. Thus, the lineage specificity of CSF-1 in hematopoiesis appears to depend on restriction of CSF-lR expression in cells of the mononuclear phagocyte lineage, rather than upon specificity in the downstream mitogenic machinery.

Attenuation of CSF-lR Kinase Activity Binding of CSF-1 to its receptor not only activates the receptor kinase, but induces internalization of receptor-ligand complexesand their rapid degradation in endosomes [86]. This process of receptor “downmodulation” limits the duration of the ligand-induced response. Under conditions in which all CSF-1binding sites have been occupied, cells remain refractory to ligand until new cell surface receptors are resynthesized. CSF-lR downmodulation requires tyrosine kinase activity, because kinase-inactivemutants altered at their ATP binding site are not degraded in response to ligand [87]. Treatment of cells with the phorbol ester 12-O-tetradecanoyl phorbol-13acetate (TPA) also leads to a rapid loss of CSF-1 binding sites [88] resulting from accelerated receptor turnover [89]. “Transmodulation” of CSF-lR by TPA and other activators of PKC differs from downregulation induced by ligand, since CSF-lR mutants lacking tyrosine b a s e activity are sensitive to TPA-induced degradation. TPA treatment was found to activate a protease that cleaves CSF-lR near its membrane-spanning segment, leading to the release of the ligand-binding domain from the cell and to the appearanceof a 50 Kd fragment representing the receptor kinase domain which was degraded intracellularly [W]. An attractive hypothesis is that transmodulationof CSF-lR by PKC is important in determining the fate of activated macrophages during the inflammatory response. Stimulation of PKC in mature mononuclear phagocytes leads not only to receptor loss, but also to destabilization of c - w mRNA [W]. These events coincide with macrophage activation associated with their increased phagocytic activity and with release of other inflammatory cytokines, including CSF-1 itself [22-251. .Transmodulationof CSF-lR would preclude an autocrine response to CSF-1 and might limit the subsequent survival of activated macrophages at sites of inflammation. Conversely, the chronicity of the inflammatory response might depend upon recruitment of naive macrophagesthat can respond to the prolikrative effects of CSF-1 released by activated cells.

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Antibody-Mediated Inhibition of CSF-lR Action Several monoclonal antibodies (MoAbs) directed to epitopes in the extracellular domain of human CSF-lR inhibit iigand binding and abrogatethe CSF-l-induced growth of receptor-bearing cells [91]. Because prebinding of CSF-1 did not reciprocally inhibit the binding of these MoAbs to CSF-lR, the antibodies appear not to react directly with epitopes within the CSF-1 binding site, but must either sterically inhibit ligand binding or decrease the receptor’s affinity for CSF-1. Intriguingly, one such antibody was also found to inhibit the ligand-independent growth of cells transformed by oncogenic c-fmr mutants, which encode receptor analogs that function constitutively as kinases (see below). When bound to CSF-lR at the cell surface, the latter MoAb did not induce receptor internalizationor degradation, suggesting that it interrupted signal transduction by affecting the conformation of CSF-lR on the plasma membrane. It remains unclear whether the confonnational changes that activate receptor kinase activity are intramolecular, or whether intermolecular associations of the receptor with itself or with other membrane proteins are required for signal transduction. Antibody-mediatedinhibition of enzymatically active CSF-lR is more consistent with the latter possibility. CSF-lR and Cell Transformation Transduction of c-fmr sequences into acutely transforming feline retroviruses, as the v-fmr oncogene, resulted in genetic alterations that constitutively activate the tyrosine kinase activity of the v-fmrcoded glycoprotein [%I. When these genetic changes were programmed into the human or feline c-fms cDNAs, their encoded products were able to induce cell transformation in the absence of CSF-1 [47,93]. The activating mutation in the human gene consisted of a single serine for leucine substitution at codon 301 in the middle of the extracellular domain, implying that the mutation mimics an effect of ligand in activating receptor kinase activity. Surprisingly, receptors containing the codon 301 mutation were able to bind CSF-1 with high affinity and were further upregulated by ligand. Substitution of only certain amino acids at codon 301 activate transformingactivity, whereas others do not [94], indicating that some specific structural requirement must be met to induce kinase activity. A possible interpretation based on studies in other receptor systems [95-981 is that a CSF-l-induced increase in the aggregation state of receptor monomers at the cell surface is a prerequisite for activating the receptor kinase and that the codon 301 mutation leads to increased dimer formation in the absence of ligand. Additional genetic changes in CSF-lR, while insufficient in themselves to induce cell transformation [50], may nevertheless contribute to oncogenicity. In particular, truncation or frame shift mutations within the receptor carboxyltermind tail, as observed in two independently transduced v-fms genes [99,1001, disrupt a negative regulatory domain and up-regulate receptor kinase activity.

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When combined with the codon 301 mutation in the human c - w gene, these carboxylterminal alterationsgenerate c-fms genes that are more potent transforming agents than receptor genes bearing the codon 301 mutation alone [93]. Thus, different mutations within the c-fms proto-oncogene can lead to a stepwise increase in basal CSF-lR kinase activity,ultimately generatingsustained signals for cell growth in the absence of ligand. Because the CSF-lR kinase can confer a ligand-dependentgrowth response in many cell types, the v-@ gene is promiscuous in its ability to alter the growth of cultured cells [92],including fibroblastsand epithelial cells [99,1001, myeloid cells [89, 1011 and pre-B lymphocytes [102]. Many such cells become independent of growth factors to which they ordinarily respond, but do not themselves produce CSF-1. For example, v-fms-infected CSF-ldependent BAC1.2F5 macrophages [89], IL3dependent FDC-PI myeloid cells [lo01or PDGFdependent NIH-3T3 fibroblasts [84] became both fixtor-independentand tumorigenicin nude mice. An interesting exception involved murine B cell precursors in long-term bone marrow culture that, while induced to proliferate to abnormally high densities, remained dependent on stromal factors and were initially non-tumorigenic [102]. With continued passage, however, factor-independenttumorigenic variants arose, suggesting that v-fms expression predisposed the cells to eventual oncogenic conversion. A similar lack of target cell specificity for transformation has been observed in vivo. The use of v-@-infected bone marrow cells to reconstitute lethally irradiated mice resulted in a variety of hematopoietic proliferative disorders, including clonal erythroleukemias and B cell lymphomas [103]. Together, these findings suggest that mutations within the c-@ gene in situ might contribute to myeloid proliferative disorders and leukemia. Using MoAbs directed to extracellular epitopes of human CSF-lR, the receptor was detected on leukemic blasts from about 40% of acute myeloid leukemia cases, whether or not they expressed other morphological, antigenic, or histochemical markers of monocytic differentiation [la]. In contrast, CSF-lR was not detected in acute lymphocytic leukemias. Polymerase chain reaction techniques can now be used to determine whether the c-fms genes in receptor-positive blast cells have been altered by critical mutations. Although initial studies will logically focus on codon 301 and the region encoding the CSF-lR carboxylterminaltail, sites of mutation other than those presently recognized might similarly lead to constitutive activation of the receptor kinase, thereby compounding the difficulty of such studies. There is also reason to suspect that inappropriateexpression of c-fms in early myeloid progenitors might predisposethem to malignant transformation.Normally, CSF-lR is first detectably expressed in immature bone marrow precursors that are committed to differentiate toward monocytes and macrophages, and the levels of receptor expression increase as the cells mature [86]. In mice, retroviral insertions 5' to the c-fins gene activate its transcription in immaturemyeloid cells, and the abnormally elevated levels of receptor expressed on these early progenitors trigger their clonal expansion and, ultimately,their leukemicconversion [105].

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Possibly, the immature cells acquire the capability to respond inappropriately to CSF-1; alternatively, overexpression of CSF-lR itself might lead to a relaxation in their growth factor requirements, providing the cells with an initial proliferative advantage. Examples of the latter phenomenon have been recently documented in model culture systems [83, 851. Although recent progress in the molecular characterization of CSF-1 and its receptor have provided a conceptual foundation and tools for future analysis, the role of ligand-receptor interactions in mediating complex biological responses in hematopoiesis, inflammation and placental development remains poorly understood. Preclinical trials with the recombinant growth factor in primates have recently indicated that CSF-1 induces monaytosis and increases antibodydependent cellular cytotoxity of macrophages [106], ameliorates the myelosuppression of chronic neutropenia of childhood [107], and may serve as a differentiating agent in the treatment of acute myelogenous leukemias [108, 1091. The use of CSF-1 congeners or antibodies to CSF-1R that inhibit ligand-receptor interactions may therefore provide novel approaches for manipulating the proliferative and functional responses of mononuclear phagocytes and their malignant counterparts.

Acknowledgments Dr.Sherr is supported by the Howard Hughes Medical Institute, grant R35-CA47064 from the National Cancer Institute, NIH, and the American Lebanese Syrian Associated Charities of St. Jude Children's Research Hospital.

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65 Orlofsky A, Stanley ER. CSF-I-induced gene expression in macrophages: dissociation from the mitogenic response. EMBO J 1987;6:2947-2952. 66 'hhinski RT, Stanley ER. The regulation of mononuclear phagocyte entry into S phase by the colony stimulating factor CSF-1. J Cell Physiol 1985;122:221-228. 67 Wahl MI, Daniel TO, Carpenter G. Antiphosphotyrosinerecovery of phospholipase C activity after EGF treatment of A431 cells. Science 1988;241:968-970. 68 Wahl MI,Nishibe S, Suh P-G, Rhee SG, Carpenter G. Epidermal growth factor stimulates tyrosine phosphorylation of phospholipase C-II independently of receptor internalizationand extracellular calcium. Proc Natl Acad Sci USA 1989;86:1568-1572. 69 Meisenhelder J, Suh P-G, Rhee SG, Hunter T. PhospholipaseC-y is a substrate for the PDGF and EGF receptor protein-tyrosine kinases in vivo and in vitro. Cell 1989; 571109-ll22. 70 Margolis B, Rhee SG,Felder S,et al. EGF induces tyrosine phosphorylation of phospholipase C-II:a potential mechanism for EGF receptor signaling. Cell 1989;57: 1101-1107. 71 Wahl MI,Olashaw NE,Nishibe S,Rhee SG. Pledger WJ,Carpenter G. Platelet-derived growth factor induces rapid and sustained tyrosine phosphorylation of phospholipase C-y in quiescent Balb/c 3T3 cells. Mol Cell Biol l989;9:2934-2943. 72 Berridge MJ. Inositol trisphosphate and diacylglycerol: Two interacting second messengers. Ann R Bioch 1987,56:159-193. 73 Downing JR, Margolis BL, Zilberstein A, et al. PhospholipaseC-y, a substrate for PDGF receptor kinase, is not phosphorylated on tyrosine during the mitogenic response to CSF-I. EMBO J 1989 (in press). 74 Whetton AD, Monk PN, Consalvey SD, Downes CP. The haemopoietic growth factors interleukin 3 and colony stimulating factor4 stimulate proliferation but do not induce inositol lipid breakdown in murine bone-marmwderivedmacrophages. EMBO J 1986;5:3281-3286. 75 Morrison DK, Kaplan DR, Roberts TM. Signal transduction from membrane to cytoplasm: growth factors and membrane-bound oncogene products increase c-mf phosphorylation and associated protein b a s e activity. P m Natl Acad Sci USA 1989;85:8855-8859. 76 Morrison DK, Kaplan DR, Escobedo JA, Rapp UR,Roberts TM, Williams LT. Direct activation of the serine/threonineb a s e activity of mf-1through tyrosine phosphorylation by the PDGF beta-receptor. Cell 1989;58:649-657. 77 RaffUR, Cleveland JL, Bonner TI, Storm SM. The mfoncogene. In: Reddy EP, Skalka AM, C u m T, eds. The Oncogene Handbook. Amsterdam-New York: Elsevier, 19883213-253. 78 Kaplan DR, Whitman M, SchaffhausenB, et al. Common elements in growth factor stimulation and oncogenic transformation: 85 kd phosphoprotein and phosphatidylinositol kinase activity. Cell 1987,50:1021-1029. 79 Auger KR,Serunian LA, Soltoff SP, Libby P, Cantley LC. PDGFdependent tyrosine phosphorylation stimulates production of novel polyphosphoinositidesin intact cells. Cell 1989;57167-175. 80 Escobedo JA, Williams LT. A PDGF receptor domain essential for mitogenesis but not for many other responses to PDGF. Nature 1988;335:85-87. 81 Das SK, Stanley ER. Structure-function studies of a colony stimulating Eactor (CSF-I). J Biol Chem 1982;25713679-13684. 82 Waheed A, Shadduck RK. Purification of colony stimulating Eactor by affiity chromatography. Blood 1982;60:238-244.

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83 Hartmann T, Seuwen K,Roussel MF, Sherr CJ. Pouyssgur J. Functional expression of the hupan receptor for colony-stimulatingfactor 1 (CSF-1) in hamster fibroblasts: CSF-1 stimulates Na'lH' exchange and DNA-synthesis in the absence of phosphoinositide breakdown. Growth Factors 1989 (in press). 84 bussel MF,Sherr CJ. Mouse NM-3'M cells expressing human CSF-1 receptors avergrow in serum-freemedium containing human CSF-1 as their only growth factor. Proc Natl Acad Sci 1989 (in press). 85 Kato J-Y, Roussel MF, Ashmun RA,Sherr CJ. Transduction of human colony stimulating factor-1 receptor into interleukin-3-dependentmouse myeloid cells induces both CSF-l-dependentand factor-independentgrowth. Mol Cell Biol W89;9:4069-4073. 86 Guilbert U,Stanley ER. The interaction of lzsI-colony-stimulatingfactor4 with bone marrow-derived macrophages. J Biol Chem 1986;261:4024-4032. 87 Downing JR, Roussel MF, Sherr CJ. Ligand and protein kinase C downmodulatethe colony-stimulating factor-1 receptor by independent mechanisms. Mol Cell Biol W89;9:2890-2896. 88 Chen BD-M, Lin HS, Hsu S. lbmor-promoting phorbol esters inhibit the binding of colony stimulatingfactor (CSF-1) to murine peritonealexudate macrophages. J Cell Physiol 1983;ll6:207-212. 89 Wheeler EF, Rettenmier CW,Look AT, Sherr CJ.The v-fmr oncogene induces factor independence and tumorigenicity in a CSF-1 dependent macrophage cell line. Nature W86;324:377-380. 90 Weber B, Horiguchi J, Luebbers R, Sherman M,Kufe D. Post-translationalstabilization of c - h mRNA by a labile protein during human monocyte differentiation. Mol Cell Biol W89;9:769-775. 91 Sherr CJ, Ashmun RA, Downing JR, et al. Inhibition of colony stimulating factor-1 activity by monoclonal antibodies to the human CSF-1 receptor. Blood 1989;73: 1786-1793. 92 Sherr CJ. Thefmr oncogene. BBA Rev Cancer 1988;948:225-243. 93 bussel MF, Downing JR, Rettenmier CW, Sherr CJ. A point mutation in the extracellular domain of the human CSF-1 receptor (c-fms proto-oncogene product) activates its transforming potential. Cell 1988;55:979-988. 94 Roussel MF, Downing JR, Sherr CJ. Transforming activities of human CSF-1 receptors with different point mutations at codon 301 in their extracellular domains. Oncogene 1989 (in press). 95 Y d e n Y,SchlessingerJ. Self-phosphorylationof epidermal growth factor receptor: evidence for a model of intermolecular allosteric activation. Biochemistry 1987;26: 1434-1442. 96 Biini-Schnetzler M, Pdch PF. Mechanism of epidermal growth factor receptor autophosphorylation and high affinity binding. Roc Natl Acad Sci USA W87,84:7832-7836 97 Weiner DB, Liu J, Cohen JA, Williams WV, Greene MI. A point mutation in the neu oncogene mimics ligand induction of receptor agpregation. Nature W89;339:230-231. 98 Heldin C-H, Ernlund A, Rorsman C, R6nnstrand L. Dimerization of B type plateletderived growth factor receptors occurs after ligand binding and is closely associated with receptor kinase activation. J Biol Chem 1989;264:8905-8912. 99 Donner L, Fedele LA, Garon CF, Anderson SJ, Sherr CJ. McDonough feline sarcoma virus: characterization of the molecularly cloned provirus and its feline oncogene (v-fms). J Virol l982;41:489-500. 100 Besmer P, Lader E,George PC,et al. A new acute transforming feline retrovirus with fms homology specifies a C-terminally truncated version of the c-fmr protein that is different from SM-feline sarcoma virus v--tins protein. J Virol 1986;60:194-203.

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101 Wheeler EF, Askew D, May S, Ihle JN, Sherr CJ. The v-fmr oncogene induces factorindependent growth and transformation of the interleukin-3-dependent myeloid cell line FDC-P1. Mol Cell Biol 1987,7:1673-1680. 102 Bonillo W, Sherr CJ.Early pre-B-cell transformation induced by the v-fmr oncogene in long-term mouse bone marrow cultures. Mol Cell Biol 1989;9:3973-3981. 103 Heard JM, Roussel MF, Rettenmier CW,Sherr CJ. Multilineage hematopoietic disorders induced by transplantation of bone marrow cells expressing the v-fmr oncogene. Cell 1987;51:663-673. 104 Ashrnun RA,Look AT, Roberts WM,et al. Monoclonal antibodies to the human CSF-1 receptor (c-fmsproto-oncogene product) detect epitopes on n o d mononuclear phagccytes and on human myeloid leukemic blast cells. Blood 1989;73:827-837. 105 Gisselbrecht S, Fichelson S, Sola B, et al. Frequent c-fmr activation by proviral insertion in mouse myeloblastic leukaemias. Nature 1987,329:259-261. 106 Garnick MB, O’Reilly RJ. Clinical promise of new hematopoietic growth factors: M-CSF, IL-3, IL-6. In: Golde DW, ed. HematologylOncology Clinics of North America: Hematopoietic Growth Factors. Philadelphia: W.B. Saunders, 1988;3:495-509. 107 Komiyama A, Ishiguro A, Kubo T, et al. Increases in neutrophil counts by purified human urinary colony-stimulatingfactor in chronic neutropenia of childhood. Blood 1988;71:41-46. 108 Miyauchi J, Kelleher CA,Wong GG, et al. The effects of combinationsof the recombinant growth factors GM-CSF, G-CSF, IL-3, and CSF-1 on leukemic blast cells in suspension culture. Leukemia 1988;2:382-387. 109 Miyauchi J, Wang C, Kelleher CA, et al. The effects of recombinant CSF-1 on the blast cells of acute myelogenous leukemia in suspension culture. J Cell Physiol 1988; 135:55-62.

Discussion Metcalf: Does the use of monomeric CSF-I lead to autophosphorylationor any evidence of aggregation? Sherr: No. Dick Stanley reported a long time ago that monomeric CSF-1 was inactive biologically. We have never retested the reduced compound for autophosphorylation. Metcalf: Are you suggesting that maybe when you downmodulate the CSF-1 receptor by an agent like GM-CSF you will also see cleavage? Sherr: We have tried both GM-CSF and IL-3 and have not seen receptor cleavage. We’ve only gotten cleavage with TPA and Di-C8, which is a more physiological and reversible activator of PKC. Lipopolysaccharide(LPS) is a very strong transmodulatorof the CSF-1 receptor response. I know Nick Nicola has reported loss of CSF-1 binding sites in response to IL-3 and GM-CSF in normal bone marmw cells, and the proteolytic degradation of CSF-1 receptors does not account for his data. Because we have not measured CSF-1 binding sites after GM-CSF or IL-3 treatment, there may be alternative mechanisms to account for a loss of binding sites in response to these hemopoietins. I’d like to emphasize, based on Dr. Griffin’s presentation before me, that the mechanism of TPA downmodulation of GM-CSF receptors in neutrophils must also be quite different, since Jim observes internalization of the ligand. One would have to assume that the GM-CSF receptor is also being internalized. With the CSF-1 receptor, the receptor is not being internalized in response to TPA, but is cleaved at the cell surface. So, I don’t think there is a unique mechanism transmodulating the expression of all these receptors.

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Widmer: Are the pre-B cells that are expressing& still sensitive to the growth-promoting effects of interleukin 7? Sherr: I can’t answer that specifically. Initially, they are stromal cell-dependent, so if dependence is mediated only by IL-7, then the answer is yes. However, we have never tested purified IL-7 for its ability to support these B cells. Moreover, the cultures rapidly generate cells that are factor-independent. In particular, when we introduced the v-& gene, the cells become rapidly independent of stromal factors. Sarmientos: Your experiments of receptor transduction are really impressive, and I have two questions on this aspect. What about the binding of IL-3 to the& oncogeneproduct? Sherr: IL-3 does not bind to the CSF-1 receptor. Sarmientos: And do you think that, technologicallyspealung, when screening for growth factor receptors, techniques like expression cloning can give you false results? Sherr: I don’t think so. I think if the experiments are done properly one can get appropriate clones. The main problem with the IL-3 and GM-CSF receptors is that there haven’t been cells that express high receptor numbers and therefore, there is probably not a lot of mRNA. Unless one utilizes a recombinant expression system, these receptors are going to be difficult to clone. Kaushansky: The question arises if FDC-PI cells synthesize CSF-l? Sherr: No, they do not.

Kaushansky: In serum-free cultures, did you use any anti-CSF-1 antibodies to make sure there wasn’t some CSF-1 responsible for the low number of background colonies? Sherr: Yes, transformation does not occur through an autocrine mechanism. There was no M-CSF message detected by northern blotting, nor was there detectable CSF-1 activity in the media. In f k t , there was nothing in the media that could support the gmwth of parental FDC-PI cells, and monoclonal antibodies directed to the receptor, which extinguished signal transduction whether CSF-1 is present or not, reduced background colony formation to zero. Therefore, I would conclude that the mechanism for factor-independentgrowth depends on constitutive receptor signals and not on endogenous growth factor production.

D’Andrea: Harvey Lodish’s lab at MIT has evidence for two different mechanisms of receptor downregulation: one involving coated pits and one not necessitating coated pits. It turns out that if you treat the A-sialoglycoprotein receptor with monoclonal antibodies, it seems to be dawnregulatedindependent of coated pits. My question is: do you have any monoclonals which downregulate c-fms independent of the TPA pathway or independent of the presumed ligand pathway? Sherr: No. We have looked at receptor turnover in cells grown in the presence or absence of monoclonals in an attempt to demonstrate accelerated degradation in response to antibodies, regardless of mechanism. We have 12 monoclonal antibodies to the human CSF-1 receptor; four of them inhibit CSF-1 binding, and one of them inhibits signal transduction even in cells that are transformed by an oncogenicfms variant. In other words, growth of transformed cells can be arrested with one, but not the other three antibodies. David Golde

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has done some work with us to show that the four antibodies that inhibit CSF-1 binding in the fibroblast system also inhibit macrophage colony foxmation from normal human bone marrow. None of the 12 antibodies induce accelerated receptor turnover. So, unlike Mark Greene’s results in which antibodies to the neu gene product induce receptor downmodulation, ours do not. Let me also emphasize that downmodulation of the CSF-1 receptor is preceded by the aggregation and cross-phosphorylation of the molecules in response to ligand. I think a dramatic example comes from an experiment that we’ve just done with Ellis Reinherz in which we’ve made a chimeric construct containing the CD2 extracellular domain fused to the kinase domain of the CSF-1 receptor. Monoclonal antibodies which aggregate CD2 and stimulate the alternative pathway in T cells aggregate this chimeric receptor in fibroblasts and induce mitogenesis. Therefore, anti-CD2 can substitute for CSF-1 in generating a full mitogenic response in this system. Our interpretation is that signal transduction is triggered through antibody-mediated receptor dimerization.

Shadduck: You mentioned that you observed clonal evolution in@-infected B cells. Did you see any selective sites for viral integration?

Sherr: No. I’ve pointed out that one of the assays for clonality, other than VDJ rearrangement which is a very good one, is to determine the sites of retroviral integration. The strategy here is that retroviruses integrate at random, and when you use restriction enzymes that don’t cut within the provirus, each integration event is characterized by a unique restriction band that hybridizes to a v-fmr probe. Dr. Gary Borzillo has analyzed more than 40 cultures now,and he has not seen any two of them in which the dominant clones have common insertion sites. I can’t formally exclude the possibility of integration in a clustered region but we have no evidence for preferred integration sites using restriction analyses.