178,

VIROLOGY

401

-409

(1990)

Transformation TERUKO instwt

TAMURA,’ ftir V/roiog/e,

of Chicken

ANGELIKA

Fibroblasts

HADWIGER-FANGMEIER,

Justus-LIebig-Universit&

Giessen, Received

March

Frankfurter

by the v-fms BRUCE

Strasse

16, 1990; accepted

BOSCHEK,

707, D-6300 May

Glessen,

Oncogene AND

Federal

HEINER

NIEMANN’

Republic

of Germany

2 7, 1990

The v-fms oncogene of the McDonough strain of feline sarcoma virus (SM-FeSV) encodes a plasma-membraneassociated tyrosine kinase (gpl40”-‘“7 which is closely related, both structurally and functionally, to the c-fms-specified receptor for the macrophage colony stimulating factor (CSF-1). In mammalian fibroblasts. the natural producers of CSF1, expression of v-fms leads to cell transformation. To study the interaction between CSF-1 and gp140V~‘mS molecules in a cell system that does not produce endogenous cross-reactive CSF-1, we have expressed the entire v-fms gene as well as a nontransforming deletion mutant (SC2) in chicken embryo cells (CEC). For this purpose the avian retroviral vectors pDS3 and PREP, based on Rous sarcoma virus, were used to isolate recombinant virus particles. CEC infected with virus that carried the entire v-fms gene expressed high amounts of gp140’-‘mS, comparable to those in SM-FeSV transformed NRK cells. However, these CEC remained flat, retained their fibronectin network, and did not produce enhanced levels of plasminogen activator. The cells grew faster than control CEC for more than 8 weeks but failed to form colonies in soft agar. Within 2 days after addition of CSF-1 to the growth medium, a transformed cell phenotype was induced, as judged by loss of the fibronectin network, again with a growth rate fourfold faster than that of the parental cells and with colony formation in soft agar. Moreover, human CSF-I caused a rapid tyrosine phosphorylation of v-fms molecules detectable within 5 min after addition of the growth factor. In contrast, CSF-I had none of the above effects on cells that expressed the SC2 v-fms deletion mutant. (~-81990 Academic Press. Inc.

INTRODUCTION The v-fms gene was detected as the transforming oncogene of the McDonough strain of feline sarcoma virus (SM-FeSV). The gene product belongs to the family of receptor tyrosine kinases (Hampe et a/., 1984). It is translated as a gaggfms fusion protein which is cotranslatronally N-glycosylated and processed by proteolytic cleavage to generate ~55~~~ and gp120”~fmS. The latter is specified entirely by v-fms and remains associated with membranes of the endoplasmic reticulum. About 10 to 15% of the gpl 20Vmfmsmolecules are transported via the Golgi apparatus to the plasma membrane and are concomitantly processed by additional glycosylation of the ammo-terminal domain to yield gp 140” fms (Anderson et al., 1984; Manger et al., 1984; Rettenmier et al., 1985). Cell surface expression of the glycoprotein is a prerequisite for cell transformation (Roussel et al., 1984). The cytoplasmrc domarn of v-fms exhibits tyrosine kinase activity. Although the detectable activity is four- to fivefold weaker than that of other viral tyrosine krnases, It IS clearly requrred for cell transformation (Tamura et al., 1986, 1988). The cellular counterpart of v-fms, c-fms, was identified as the receptor for the macrophage colony stimulating factor (CSF-1) (Sherr et a/., 1985). Since fibro’ To whom addressed 2 lnstltut

krankhelten Tijbtngen,

requests

for

reprrnts

ftir Mlkroblologle. der Tlere, Postfach FRG

and

correspondence

Bundesforschungsanstalt 1149, Paul-Ehrlich-Strasse

should fijr

be Vlrus~ 28, 74

blasts are the natural producers of CSF-1, it was initially suggested that the preserlce of CSF-1 contributed to v-fms-induced cell transformation (Sherr et al., 1985). Furthermore, the receptor-binding domains of CSF 1 from various mammalian species are highly conserved at the amino acid level. From 8 1 to 88% homology has been demonstrated between human, mouse, and feline CSF-1 molecules (Kawasaki et al., 1985; Wong et a/., 1987, DeLamarter et a/., 1987; Ladner et a/., 1988; Hadwiger-Fangmeier and Tamura, manuscript in preparation). However, the sequences of the extracellular factor-binding domains of the CSF-1 receptors diverge more dramatically between various species. Although expression of the v-fms gene in mouse fibroblasts leads to cell transformation, murine CSF-1 was shown to bind only weakly to cell-surface-expressed v-fms molecules (Sacca et al., 1986). indicating a nonautocnne mechanism of transformation. An autocrine mode of transformation may be assumed in those instances in which fibroblasts simultaneously express the CSF-1 receptor and a cross-reactive or homologous CSF-1, as observed in the munne system (RohrSchneider et a/., 1989; Lyman et al., 1988). To study the interaction of CSF-1 with v-fms in a cell system that is free of potentially interfering mammalian CSF-1 s, we have now expressed the v-fms gene in CEC. MATERIALS AND METHODS Cell and virus Fertilized chicken eggs (C/O) were supplied by Lohmann (Cuxhaven, FRG). Primary CEC were prepared

TAMURA

402

and infected after the first passage with virus according to published procedures (Friis et al., 1975). Cells were grown in Dulbecco’s modified Eagle’s minimum essential medium (DMEM) supplemented with 10% fetal calf serum. Antisera Anti-v-fms antiserum was prepared in rabbits using an MS2-v-fms fusion protein expressed from a recombinant pEX31 b vector in Escherichia co/i as described previously (Tamura et al., 1989). Antiserum against the Prague B strain of Rous sarcoma virus was prepared as described (Hadwiger and Bosch, 1985). lmmunofluorescence

techniques

The procedures have been described mura et al., 1988). Plasmid

constructions

and transfection

previously

(Ta-

procedure

PREP (a kind gift from H. Hanafusa, Rockefeller Institute, NY) contained one LTR and the gag, pol, and the 5’-portion of the env genes of Rous sarcoma virus (Cross and Hanafusa, 1983). pDS3 was kindly provided by Dr. Iba (Institute of Medical Science, University of Tokyo, Japan). This vector is derived from pN4 and pXD-Rl 13 (Hanafusa et al., 1984; Kornbluth et al., 1986) and lacks the entire v-src gene. The gag and pol sequences were deleted by cleavage with Xhol and religation. A singularBg/II site served as the recipient site for individual v-fms constructs. The SM-FeSV genome present in pBRSM-FeSV was a kind gift from C. Sherr, St. Jude Children’s Hospital (Memphis, TN). The singular /Vhel site (position 2326) was filled in with Klenow polymerase and a BamHl linker was inserted. A deletion of amino acids 91 to 188 within the N-terminal extracellular domain of v-fms was generated by double digestion of the SM-FeSV genome with Smal (cleaving in position 2667) and C/al (position 2959) a fill-in reaction with Klenow polymerase, and subsequent religation. The two v-fms-specific BamHl fragments, each containing 72 5’-noncoding and 554 3’-noncoding nucleotides, were cloned into the Bglll site of pDS3 to yield pNB4 and pSC2, respectively. DNA transfections were carried out essentially as described by Cross and Hanafusa (1983). For this purpose pDS3, pNB4, and pSC2 were completely digested with SalI and were ligated with Sa/l-digested PREP. Radiolabeling

of CEC

Subconfluent CEC infected with virus in 5- or 1 O-cm petri dishes were labeled over 6 hr with 100 &i/ml of L3H]leucine (55 Ci/mmol), [6-3H]glucosamine hydro-

ET AL.

chloride (37 Ci/mmol), [35S]methionine, or 1 mCi/ml of [32P]orthophosphate (1,850 MBq, carrier-free). All radioisotopes were from Amersham Buchler. Immunoprecipitation, kinase assay, phosphoamino acid analysis These assays were performed viously (Tamura et al., 1988).

and as described

pre-

RESULTS Construction of a retroviral vector for the expression of the v-fms oncogene in chicken cells Infectious Rous sarcoma virus (RSV) can be recovered by introducing intact RSV proviral DNA into CEC (Hill and Hillova, 1972). Together, plasmids PREP and pDS3 (Cross and Hanafusa, 1983; Iba et al., 1988) provide all of the information for a replication-competent virus (Fig. 1). PREP (containing one LTR, gag, pol, and the 5’-portion of env) and pDS3 (containing the 3’-portion of env, the splice acceptor site for the smallest subgenomic mRNA in RSV and one I&$ were ligated at their Sa/l sites and the products wer&ansfected into CEC (Cross and Hanafusa, 1983; Iba et al., 1988). Since the v-fms peptide by itself provides a signal sequence for integration into the RER, the amino-terminal gag portion may be deleted without affecting membrane insertion and the subsequent conversion into gp140V’mS during transport to the plasma membrane (Wheeler et al., 1986a). Roussel et al. (1987) have demonstrated that constructs lacking all gag sequences transformed NIH 3T3 cells as efficiently as SM-FeSV. We have repeated such studies in NRK cells using a mutant SM-FeSV from which the entire gag portion had been deleted between the Sstl site (located in position 641 within the 5’-noncoding leader sequence of the gag gene) and the singular Nhel site (position 2326, 72 nucleotides upstream from the ATG translation start codon of the vfms gene). In agreement with previous data (Wheeler et al., 1986a; Roussel et al., 1987), this deletion did not reduce the transformation capacity of v-fms in NRK cells. Therefore, we have cloned the v-fms gene from the Nhel site to the BamHl site into the Bglll recipient site of pDS3 to yield pNB4 (Fig. 1). A similar construct, designated pSC2, was made with a mutant v-fms gene carrying a deletion of 291 nucleotides corresponding to amino acids 91 to 188 within the extracellular factorbinding domain (for details, see the legend to Fig. 1 and Materials and Methods). CEC were transfected with concatamers obtained by ligation of Sail-digested pNB4, pSC2, or pDS3 (as controls) with PREP DNA linearized with the same enzyme (Iba et a/., 1988). Transfected CEC were subcul-

THE

vfms/CSFF

SYSTEM

IN CEC

403

S

PDS3

PREP-derived

PNS~-

derlvatlves

or pSC2-derived

FIG. 1. Constructron of retrovrral vectors for the expressron of v-fms genes in CEC. The followrng restrictron endonucleases were used: B, BarnHI, Bg, Bglll, C, C/al; N, iVhel; S, %/I; Sm, Smal; X, Xhol For detarls of the orrgrn of PREP and pDS3, see Materials and Methods. The SM FeSV genome contarnrng two LTRs (striped boxes) and the vfms gene (dotted box) are shown at the upper rrght. After conversron of the lVhel site Into a BamHl site and a Smal-C/al deletion the individual vfms-specific BarnHI fragments were cloned into the Bgill recipient sate of pDS3 to yield pNB4 or pSC2. The RSV-derived LTRs are rndrcated by solid boxes. Concatamenc DNA, rn some Instances contarnrng the gag, pal. and env genes of RSV In the desired orrentatron. was prepared by digestion of PREP and pNB4 or pSC2 DNA with Sail and subsequent Irgation.

tured on two separate plates. One plate contained 0.4% soft agar for the detection of foci, while the other was incubated with fluid growth medium to harvest recombinant virus strains, designated DS3, NB4, and SC2. No clear foci and no morphological changes were detected within 2 weeks in CEC transfected with any of the above constructs. The gag and env proteins of RSV, however, were immunoprecipitable from the individual culture supernatants throughout this time period, indicating that viral replication took place (data not shown). Synthesis, glycosylation, and phosphorylation of v-fms molecules in virus-infected CEC To determine whether DS3-, NB4-, or SC2-infected CEC (DS3-, NB4-, or SC2-CEC) expressed v-fms-specific polypeptides, cultures were metabolically labeled with [3H]leucine, [6-3H]glucosamine, or [3’P]orthophosphate over 6 hr and cell lysates were subjected to immunoprecipitation. Since the SC2-specified fms proteins were neither immunoprecipitable nor indirectly labeled in immunofluorescence studies by any of

our rat antisera raised against SM-FeSV-transformed NRK cells, we employed a polyclonal anti-fms rabbit an tibody that was raised against a bacterially expressed v-fms fusion protein. This antibody was shown to recognize the cytoplasmic portion of the v-fms molecules (Tamura et a/., 1989). As shown in lanes 2 of Figs. 2a and 2b, gp120”~‘ms and gp140”~‘m” were synthesized and glycosylated in NB4-CEC in proportions similar to those observed in mammalian cells (Manger et a/., 1984; Rettenmier et al., 1985). As reported previously (Tamura et al., 1986), It IS the gp140V~fmS molecules present in the plasma membrane of fms-transformed normal rat kidney (NRK) cells that are predominantly phosphorylated at serine, threonine, and tyrosine residues In NB4-infected CEC, gpl 40V~fmswas again detected as the major phosphoprotein (Fig. 2c, lane 2) containing, however, only phosphoserine and phosphothreonine (see Fig. 6). Taken together, our data demonstrate that, with the exception of tyrosine phos phorylation, apparently all the other essential brosynthetic steps involved in the formation of functional gp 14ovmfmsmolecules in the plasma membrane occur in

404

AURA

a

C

b

66-

1

2

3

1

2

3

12

3

glycosylation, and phosphorylation of v-fms molFIG. 2. Synthesis, ecules in CEC infected with recombinant virus. CEC infected with DS3 (lanes l), NB4 (lanes 2) and SC2 (lanes 3) were labeled with [3H]leucine (a), [6-3H]glucosamine (b). or [32P]orthophosphate (c) over 6 h. Cell lysates were subjected to immunoprecipitation using anti-fms rabbit serum. The products were analyzed on a 7.5% SDSpolyacrylamide gel.

NB4-CEC with characteristics similar to those observed in mammalian cells. In SC2-CEC, only a single truncated glycoprotein species, designated gpl 04A’mS, was detected after labeling with [3H]leucine or [3H]glucosamine. Indirect immunofluorescence studies on fixed cells with an antibody against the cytoplasmic domain of v-fms indicated that the intracellular distribution of this mutant molecule involved the RER and the Golgi. This distribution was indistinguishable from that of the wild-type vfms polypeptides (data not shown). We were unable to directly demonstrate a cell surface expression of this mutant v-fms polypeptide by surface labeling because none of our anti-fms sera recognized the truncated extracellular domain of v-fms. However, since the majority (>90%) of the carbohydrate side chains of gp104A-fm5, in contrast to those of gpl 40Vrms, were sensitive to treatment with endoglucosaminidase H (data not shown), it is unlikely that this molecular species is indeed expressed at the plasma membrane of SC2CEC. In agreement with this conclusion, we were unable to detect any in viva phosphorylation of gp 104A-fmS In SC2CEC (Fig. 2c, lane 3) indicating that these phosphorylation steps take place only after the fms molecules have reached the plasma membrane (Tamura et al., 1986). Nevertheless, SC2-CEC were included as control cells in these studies, since they expressed a v-fms-specific polypeptide that was associated with internal membranes and contained a potentially functional tyrosine kinase activity, as demonstrated by in vitro assays (see below). To correlate the levels of v-fms expression in NB4and SC2-CEC with those in SM-FeSV-transformed NRK cells, we performed tyrosine kinase assays on im-

ET AL

munoprecipitates of cell lysates derived from 10” cells each. Estimations based on metabolic labeling experiments would have given misleading values due to the different metabolic activities of the nontransformed CEC and the transformed NRK cells. As shown in lane C of Fig. 3, SM-FeSV NRK cells contained small amounts of the gag-fms precursor gpl 80gagmv-fms,the intracellular form gpl 20Vefms, and the terminally glycosylated mature gpl 40Vmfms,known to be expressed at the plasma membrane (Tamura et a/., 1986). NB4-CEC (lane 2) contained gpl 40V-fmSand gpl 20Vtms in similar amounts and their constant ratio reflected similar processing as in SM-FeSV NRK cells. In contrast, SC2-CEC (lane 3) contained reduced amounts of gpl 04A-fmS(lane 3), indicating either that the expression of the deletion mutant was weaker than the wild-type gene or that its tyrosine kinase activity was somewhat impaired. The addition of human CSF-1 to either of these in vitro kinase reactions had no influence on the degree of autophosphorylation even when such reactions were performed for only 5 min and the reaction temperature was reduced to 10” (data not shown).

Expression of v-fms in CEC increases the growth rate but does not lead to full cell transformation Ten days after infection of CEC with NB4, SC2, or DS3, cells contained similar levels of RSV-specific structural proteins, as judged by indirect immunofluorescence labeling. To compare the growth rates, sister cultures, each containing 5 X 1 O4 cells infected with either NB4, SC2, or DS3, were freshly prepared 2 weeks after infection. Viable cell counts were determined at the time intervals indicated from triplicate cultures (Table 1). NB4-CEC grew 1.6- to 1.7-fold faster than SC2- or DS3-infected negative control CEC. After 4 weeks of

kd 205-

66-

C

12

3

FIG. 3. ln vitro kinase activity of v-fms polypeptides. Cell lysates from 1 O6 SM-FeSV-transformed NRK cells (lane c), DSB-CEC (lane I), NB4-CEC (lane 2) or SC2-CEC (lane 3) were subjected to rmmunoprecipitation using anti-fms rabbit serum. Kinase activity was determined by the additron of [T-~‘P]ATP to the immune complexes. Products were analyzed on a 7.5% SDS-polyacrylamide gel.

THE TABLE DOUBLING

1

TIME OF CEC INFECTED WITH NB4, SC2, OR DS3 Doubling

Weeks after Infection 2 3 4 8

v-fms/CSF-1

NB4-CEC 23 35 52.5 96

trmes

SYSTEM

IN CEC

405

4G). In addition, NB4- and SC2-CEC failed to grow in soft agar (Fig. 5, left column) or to produce elevated levels of plasminogen activator (Table 2).

(h)

SC2-CEC

DS3mCEC

37 5 61 144 No growth

39 56 128 No growth

/Vote. Cells were seeded In duplrcate plates In complete medium contarnrng 10% fetal calf serum at 5 x 1 O4 cells per 50.mm-diameter culture dish. Vrable cell counts were determined from triplicate cultures harvested. Doubling times were calculated from cell counts averaged from three separate experiments.

incubation noninfected and SC2-infected CEC were almost unable to grow, while NB4-CEC were capable of growing up to 8 weeks. However, as will be discussed in more detail in the next section, none of the infected cultures became transformed, as judged by cell morphology (compare Figs. 4A and 4E) and the continuous presence of fibronectin network (compare Figs. 4C and

The addition of human CSF-1 to v-fms expressing CEC causes full cell transformation To assess whether the incapability of v-fms to transform CEC could be overcome by the addition of exogenous CSF-1, we reanalyzed the above transformationspecific parameters of NB4-, SC2-, and DS3-CEC after the addition of human CSF-1 to the growth medium. As shown in Fig. 4 (compare Figs. 4A and 4B), the addition of CSF-1 induced a fusiform cell morphology of NB4CEC within 2 days after treatment, while having no effect on the morphology of DS3-CEC (compare Figs. 4E and 4F) or SC2-CEC (data not shown). Furthermore, upon stimulation with CSF-1, NB4-CEC lost the fibronectin network within 24 h (compare Figs. 4C and 4D), grew again about twofold faster than nontreated NB4CEC, and secreted elevated levels of plasminogen activator (Table 2). Interestingly, the doubling time of CSF1 -treated NB4-CEC returned to normal values withrn 3 days upon removal of CSF-1 from the growth medium,

+

NB4CEC

DS3CEC

FIG. 4. Influence of CSF-1 on the morphology of NB4-CEC (A-D) or DSB-CEC (EEH). Cells were replated 15 days after infection using medium wrth 1000 U/ml of human CSF-1 (+) or no CSF-1 (-). (A, B, E, and F) Cells were fixed 2 days after berng replated and visualrzed by Gremsa staining. (C, D, G, and H)The dlstnbution of flbronectin was determined by indlrect lmmunofluorescence labeling. The bars represent 5 pm.

406

TAMURA

ET AL.

CSF-1

2

FIG. 5. Growth of NB4- and SCZ-CEC in soft agar. NB4-infected (panel 5% of fetal calf serum in the presence or absence of 1000 U/ml of human days. The bar represents 100 Frn

indicating that the CSF-l-induced stimulation was reversible. In addition, the panel 1 of Fig. 5 clearly demonstrates that the addition of CSF-1 allowed NB4-CEC to form colonies in soft agar. As shown in the control panels of Figs. 4 and 5 and in Table 2, none of these alterations was induced by CSF-1 in SC2- or DS3-CEC. Taken together, our findings indicate that transformation of CEC by v-fms requires the continuous presence of CSF-1. CSF-1 induces tyrosine phosphorylation internalization of gp140”~fms in NB4-CEC

and

In order to determine whether CSF-1 treatment altered the precursor/product relationship of the individual v-fms proteins, NB4-CEC were labeled over 7 h with [3H]leucine in the presence of 1000 U/ml of CSF-1. The labeled cells were subjected to v-fms protein-specific immunoprecipitation. In agreement with published data (Roussel el al., 1987) the amount of gp140”-fmS, but not that of gp120”-fms, was reduced by treatment with CSF-1 (Fig. 6a). This reduction of gpl 40Vrms molecules has been ascribed to an internalization and subsequent degradation of gpl 40V-fms polypeptides (Roussel et al., 1987). To determine whether the CSF-1 -mediated conversion to the transformed phenotype was accompanied

1) or SC2-infected CEC (panel 2) were grown in 0.4% agar containing CSF-1. All photographs were taken at identical magnificatton after 10

by an activation of the v-fms-specific tyrosine kinase activity in NB4-CEC, we prelabeled cells over 6 h with [32P]orthophosphate prior to a 5min stimulation with 1000 U/ml of human CSF-1. Cell lysates were immunoprecipitated with anti-v-fms antibody. The total phos,phorylation of the v-fms molecules was not changed (Fig. 6b, compare lanes 1 and 2). Phosphoamino acid analyses were performed on gp140”.‘“” material derived from unstimulated CEC (Fig. 6c, panel l), and CSF-l-stimulated CEC (Fig. 6c, panel 2). Only phosphoserine and phosphothreonine were detected in material from nonstimulated cells, while gpl 40”-fms from CSF-1 -treated cells clearly contained phosphotyrosine in addition to the former phosphoamino acids. From these data we conclude that exogenously added CSF-1 induces a rapid activation of the v-fms-specified tyrosine kinase and consequently an activation of the cascade leading ultimately to cell transformation. As expected, SC2-CEC expressing the mutant v-fms polypeptide showed no phosphorylation of gpl 04A-fms regardless of whether CSF-1 was present in the growth medium or not (Fig. 6b, lanes 3 and 4). DISCUSSION The transformation of mammalian fibroblasts through the viral fms oncogene is initiated by the surface ex-

THE

v-fms/CSF-1

SYSTEM TABLE

EFFECTS

OF CSF-1

ON THE TRANSFORMATION

IN CEC

2

PARAMETERS

OF CEC INFECTED

NB4-CEC

Doublrng time (h)” Degradatron of frbronectrn Casernolysis’ Colony formatron in 0.4% Actrn cables’

+CSF-1

23

15 ++ + +t tipd

agar +

WITH NB4,

SC2,

OR DS3

SC2-CEC

CSF-1

network’

407

CSF-1

DS3-CEC +CSF-1

CSF- t

37 5

38

39

38

t

t

in

b

iVote. All transformatron-specrfrc parameters were measured 2 weeks after Infection. a Doubling times were calculated from cell counts averaged from three separate experrments. b Fibronectrn and actrn cable were measured by rndrrect rmmunofluorescence. Actrn cables were observed reproducibly In more the cells ’ Cells were grown as monolayers under DMEM contarnrng 0.4% agar, 2% nonfat dry milk. and 1% chicken serum In the presence of 1000 U/ml of CSF-1 (Goldberg, 1974; Beug and Graf, 1980) d In some cells a reduction of actin cable and the formation of ruffles along the border lines of NWCEC was observed

pression of a glycoprotein, gpl 40V-fms, that functions as an activated receptor for the growth factor CSF-1. Several lines of evidence indicate that the v-fms-specific transformation mechanism of mammalian fibroblasts may in some instances be exclusively nonautocrine while in other instances it could have an additional autocrine component. This is best illustrated by the observatron that mutational activations of the cellularfmsreceptor kinase can be further enhanced by exogenous CSF-1 (Roussel er al., 1988; Roussel and Sherr, 1989)

a

6645-

b

2 p--5

FIG. 6. Effect of CSF-1 on the tyrosine phosphorylatron of v-fms polypeptrdes. (a) NB4-CEC were labeled with [3H]leucine for 7 h In the absence (lane 1) or the presence (lane 2) of 1000 U/ml of human CSF-1. Cell lysates were subjected to fms-specrfrc rmmunopreciprtatlon and SDS- polyacrylamide gel electrophoresis. (b) NB4-CEC (lanes 1 and 2) or X2-CEC (lanes 3 and 4) were labeled with [3*P]orthophosphate for 6 h and were then incubated for 5 mm In medium wrth (lanes 2 and 4) or without 1000 U/ml of human CSF-1 (lanes 1 and 3) and analyzed as in (a). (c)The gpl 40Vtms bands from untreated [panel 1, (b) lane l] and CSF-1 -treated cells [panel 2, (b) lane 21 were excised from the gels and phosphoamrno acid analyses were performed: p-S (phosphoserine), p-T (phosphothreonrne), p-Y (phosphotyrosine).

+CSF-

1

than 80% of or absence

The scope of this work was to establish a system that allowed the investigation of interactions between the v-fms oncogene product with various exogenous CSF-1 molecules in the absence of potentially interfering mammalian CSF-1 species. Therefore, we have now expressed the v-fms gene from a recombinant Rous sarcoma virus in primary chicken fibroblasts that lack a cross-reactive endogenous CSF-1. Apparently, expression of the fms-gene in these cells follows the same principles as in mammalian fibroblasts. Thus, similar expression levels and similar ratios of gpl 20Vfmsto gp140” fms molecules were observed In both cell types (Fig. 3) indicating that intracellular processing and efficiency of transport of gp 140” fmsto the plasma membrane were not altered in CEC. Our data, however, clearly demonstrate that CEC retain their normal phenotype despite gp140”~fmSlevels equivalent to those capable of transforming NRK cells. In keeping with this observation, we were unable to demonstrate any in viva tyrosine kinase activity and gpl 40vfrns molecules lacked detectable amounts of phosphotyrosine. We have to assume, however, that the low levels of tyrosine kinase activity are sufficrent to promote the observed doubling of the growth rate and the concomitant Increase in the life span of the primary cells. Despite these altered growth properties, no changes in transformation-specific parameters such as the degradation of fibronectin network, the release of plasminogen activator, cell morphology or growth in soft agar, were observed (Table 2). Recently, Fuhrmann et a/. (1989) observed a very weak v-fms-specific transformation of CEC even in the absence of exogenous CSF-1 (involving 50% loss of

408

TAMURA

actin cables, a slight reduction of fibronectin network, and a twofold increase in hexose uptake). However, these features, induced by a similarv-fms recombinant virus, were obtained in total primary CEC which apparently were not passaged prior to infection (H. Beug, personal communication). In our experiments CEC were infected only after two passages, i.e., when predominantly fibroblasts were present in the culture. Furthermore, we tested the construct described by Fuhrmann et al. (1989) in our cell system according to our protocol. The results obtained were identical to those described in this study for our viral construct. Again we found that CSF-1 induced a rapid alteration of the transformation-specific parameters (T. Tamura and H. Beug, unpublished). Transition of the cells into the transformed phenotype required the addition of exogenous CSF-1. We report here that the addition of human CSF-1 to v-fmsexpressing CEC causes an additional growth stimulation, leads to a rapid tyrosine phosphorylation of the receptor within 5 min, and induces alterations of several transformation-specific parameters such as the degradation of fibronectin network, the release of enhanced levels of plasminogen activator, fusiform cell morphology, and growth in soft agar (Table 2). The depolymerization of actin cable, however, was seen only in about 50% of the cells. In these instances the formation of ruffles along the cell borders of NB4CEC was observed. In this respect v-fms-expressing CEC behave like SM-FeSV-transformed mink cells or SMFeSV-transformed primary cat fibroblasts, both of which still retain actin cables (Anderson et a/., 1984; our own unpublished data). It is possible that the addition of CSF-1, a homodimer, will induce dimerization of the gp140”‘mS molecules exposed at the plasma membrane of NB4-CEC. A similar activation mechanism has been proposed for the EGF receptor (Yarden and Schlessinger, 1987). We further postulate that this aggregation, which appears to be a prerequisite for the activation of the cytoplasmic tyrosine kinase, can also occur even in the absence of CSF-1, albeit at a reduced rate. Therefore, CSF-lindependent cell growth may be observed in those instances when very high levels of v-fms are expressed, as has been reported for the macrophage cell line BAC1 infected with a retrovirus carrying the v-fms gene (Wheeler et a/., 1986b). Our finding that the addition of exogenous CSF-1 to v-fms-expressing CEC leads to a rapid tyrosine phosphorylation and subsequent cell transformation further implies that chicken cells provide an ideal system to further characterize putative target proteins that participate in the signal transduction cascade initiated by the binding of CSF-1. Apparently, this chain of events occurs in CEC in a similar manner to that observed in

ET AL.

mammalian cells. Coexpression of the recently cloned feline CSF-1 gene will allow the characterization of such target proteins.

ACKNOWLEDGMENTS We thank Heike Tributh and Sigrun Broehl for excellent technical assistance and Rudolf Rott for supporting this project. This work was supported by the Deutsche Forschungsgemeinschaft (Ta-1 1 l/l/l).

REFERENCES ANDERSON, S. J., FURTH, M., WOLFF, L., RUSCE~I, S. K., and SHERR, C. J. (1984). Subcellular localization of glycoproteins encoded by the viral oncogene v-fms. /. Viral. 44, 696-702. BEUG, H.. and GRAF, T. (1980). Transformation parameters of chicken embryo fibroblasts infected with the ts34 mutant of avian erythroblastosis virus. Virology 100, 348-356. CROSS, F. R., and HANAFUSA, H. (1983). Local mutagenesis of Rouse sarcoma viruses: The major sites of tyrosine and serine phosphorylation are dispensable for transformation. Cell34, 597-608. DELAMARTER, J. F., HESSION, C., SEMON. D., GOUGH, N. M., ROTHENBUHLER, R., and MERMOD, J.-J. (1987). Nucleotide sequence of a cDNA encoding murine CSF-1 (Macrophage-CSF). Nucleic Acid. Res. 15,2389-2390. FRIIS, R. R., MASON, W. S.. CHEN. Y. L., and HALPERN, M. S. (1975). A replrcation defective mutant of Rous sarcoma virus which fails to make a functional reverse transcriptase. Virology 64,49-62. FUHRMANN, U., VENNSTRBM, B.. and BEUG, H. (1989). The mutated, myeloid cell-specific growth factor receptor v-fms transforms avian erythroid but not myeloid cells Genes. Dev. 3, 2072-2082. GOLDBERG, A. R. (1974). Increased protease levels in transformed cells: A casein overlay assay for the detection of plasminogen activator production. Ce//2, 95-102. HAMPE, A., GOBET. M., SHERR, J., and GALIBERT, F. (1984). Nucleotide sequence of the feline retroviral oncogene v-fms shows unexpected homology with oncogenes encoding tyrosine-specific protein krnases. Proc. Nat/. Acad. Sci. USA 81, 85-89. HADWIGER, A., and BOSCH, J. V. (1985). Characterization of the endogenous retroviral envelope glycoproteins found in the sera ev3 and ev6 chickens. J. Gen. Viol. 66, 2051-2056. HANAFUSA, H., IBA. H., TAKEYA, T., and CROSS, F. R. (1984). Transforming activity of c-src gene. ln “Cancer Cell 2.” pp. 1-7. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. HILL, M., and HILLOVA, J. (1972). Virus recovery in chicken cells tested with Rous sarcoma cell DNA. Nature, NewBiol. 237, 35-39. IBA, H., SHINDO, Y., NISHINA, H., and YOSHIDA, T. (1988). Transforming potential and growth stimulating activity of the v-fos and c-fos genes carried by avian retrovirus vectors. Oncogene Res. 2, 12 l133. KAWASAKI, E. S., LADNER, M. B., WANG, A. M., ARSDELL, J. V., WARREN, M. K.. COYNE, M. Y., SCHWEICKART, V. L., LEE, M.-T., WILSON, K. J., BOOSMAN, A., STANLEY, E. R., RALPH, P.. and MARK, D. F. (1985). Molecular cloning of a complementary DNA encoding human macrophage specific colony stimulating factor (CSF-1). Science 230,291-296. KORNBLUTH, S., CROSS, F. R., HARBISON, M., and HANAFUSA, H. (1986). Transformation of chicken embryo fibroblasts and tumor induction by the middle T antigen of polyomavirus carried in an avian retroviral vector. Mol. Cell. Biol. 6, 1545-l 551. LADNER, M. B.. MARTIN, G. A., NOBLE, J. A., WITMAN, V. P., WARREN, M. K., MCGROGAN, M., and STANLEY, E. R. (1988). cDNA cloning and expression of murine macrophage colony-stimulating factor from L929 cells. Proc. Nad. Acad. SC;. USA 85. 6706-6710.

THE

v-fms/CSF-1

LYMAN, S. D., PARK, L., and ROHRSCHNEIDER, L. R. (1988). Colony stemulating factor-l induced growth stimulation of v-fms transformed flbroblasts Oncogene 3,391-395. MANGER, R., NAJITA, L., NICHOLS, E. J., HAKOMORI, S.-l., and ROHRSCHNEIDER, L. R. (1984). Cell surface expresslon of the McDonough strain of feline sarcoma virus fms gene product (gp140’ms). Ceil39, 327-337. RETTENMIER. C. W., ROUSSEL, M. F., QUINN, C. O., KITCHINGMAN, G. R., LOOK, A. T., and SHERR, C. J. (1985). Transmembrane onentatlon of glycoproteins encoded by the v-fms oncogene. Cell 40, 971~ 981 ROHRSCHNEIDER, L. R., ROTHWELL. V M., and NICOLA, N. A. (1989). Transformation of murine flbroblasts by a retrovirus encoding the murine c-fms proto-oncogene. Oncogene 4, 1015-l 022. RoussEL, M. F., and SHERR, C. 1. (1989). Mouse NIH 3T3 cells expressing human colony-stimulating factor 1 (CSF-1) receptors overgrow In serum-free medium contalnlng human CSF-1 as their only growth factor. froc. Nat/. Acad. Sci. USA 86, 7924-7927. ROUSSEL, M. F., DULL, T. J., RETTENMIER, C W., RALPH, P., ULLRICH, A and SHERR, C. 1. (1987). Transforming potential of the c-fms proto-oncogene (CSF-1 receptor) Rlature (London) 325, 549-552. ROUSSEL, M. F , RETTENMIER, C. W., LOOK, A. T., and SHERR, C. J. (1984). Cell surface expression of v-fms-coded glycoproteins IS requlred for transformatton. Mol. Celi. Biol. 4, 1999-2009. ROUSSEL, M. F., DOWNING, J. R., RE~TENMIER, C. W., and SHERR, C. J. (1988) A point mutation In the extracellular domaln of the human CSF-1 receptor(c-fms proto-oncogene product) activates its transforming potential. Cell55, 979-988. SACCA, R., STANLEY, E. R , SHERR. C 1.. and RETTENMIER, C. W. (1986). Specific bIndIng of the mononuclear phagocyte colony-stimulating factor CSF-1 to the product of the v-fms oncogene. froc. Nat/. Acad. SC/ USA 83,33313335. SHERR. C J , RETTENMIER. C W., SACCA, R., ROUSSEL, M. F., LOOK, A T , and STANLEY, E. R. (1985). The c-fms proto-oncogene prod-

SYSTEM

IN CEC

409

uct IS related to the receptor for the mononuclear phagocyte growth factor, CSF-1. Ce//41,665&676. TAMURA, T., SIMON, E., NIEMANN, H.. SNOEK, G. T.. and BAUER, H. (1986). gp140Yfms molecules expressed at the surface of cells transformed by the McDonough strain of feline sarcoma virus are phosphorylated In tyrosine and serine. Mol. Celi. Biol. 6, 4745 4748. TAMURA, T., HENNIG, D., GRELL. M , NIEMANN, H , and BOSCH~~K, B. (1988). Isolation of a transformation-defective mutant of the McDonough strain of feline sarcoma virus exhlbitlng tyroslne klnase activity in vitro but not in VIVO. J. I/iroi. 62, 2150 21 57. TAMURA, T., BROST, H., K&ISCH, A, IAMP~RT. F , HADWIGER-FANG MEIER, A., and NIEMANN, H. (1989). Detection of fmsoncogenespecific tyrosine kinase actlvtty In human leukemia cells. Cancer Res. C/in. Oncoi. 115, 235-241. WHEELER, E. F , ROUSSEL, M. F.. HAMPE, A WALKER, M. H., FRIEL), V. A., LOOK, A. T., RE~ENMIER. C. W , and SHERR, C. J. (1986a). The amino-terminal domain of the v-fms oncogene product Includes a functional signal peptide that directs synthesis of a transforming glycoproteln In the absence of feline leukemia virus gag sequence. J. Hrol. 59, 224-233. WHEELER, E. F RETTENMIER, C W.. LOOK, A. -; , and SHERR, C. J. (1986b). The v-fms oncogene Induces factor Independence and tumorgenlclty In CSF-1 dependent macrophage cell Ilne. /Varure (London) 324, 377 -380. WONG. G G., TEMPLE, P A., LEAKY, A C., WITEK-GIANNOTT, J. S , YANG, Y.-C., CIARLE~A, A. B., CHUNG. M MURTWA, P , KRIZ. R , KAUFMAN, R. J.. FERENZ. C R SIBLEY, B S TURNER. K. J., HEWICK, R M., CLARK, S C., YANAI, N.. YOKOTA. H.. YAMADA, M., SAITO, M , MOTOYOSHI, K , and TAKAKU, F. (1987). Human CSF-1 molecular cloning and expression of 4 kb cDNA encodlng the human urinary protein. Science 325, 1504-l 508. YARDEN, Y., and SCHLESSINGER, 1. (1987). Epldermal growth factor Induces rapid, reversible aggregation of the purified epldermal growth factor receptor. B/ochem,srry 26, 1443 145 1

Transformation of chicken fibroblasts by the v-fms oncogene.

The v-fms oncogene of the McDonough strain of feline sarcoma virus (SM-FeSV) encodes a plasma-membrane-associated tyrosine kinase (gp140v-fms) which i...
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